WO2021004838A2 - Rubisco activase with reduced adp inhibition and uses thereof - Google Patents

Rubisco activase with reduced adp inhibition and uses thereof Download PDF

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WO2021004838A2
WO2021004838A2 PCT/EP2020/068385 EP2020068385W WO2021004838A2 WO 2021004838 A2 WO2021004838 A2 WO 2021004838A2 EP 2020068385 W EP2020068385 W EP 2020068385W WO 2021004838 A2 WO2021004838 A2 WO 2021004838A2
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allele
knock
subgenome
out mutant
mutant rca
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PCT/EP2020/068385
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French (fr)
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WO2021004838A3 (en
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Andrew SCAFARO
Alexander Galle
David DE VLEESSCHAUWER
Matthew Hannah
Jeroen Van Rie
Iris FINKEMEIER
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BASF Agricultural Solutions Seed US LLC
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication of WO2021004838A2 publication Critical patent/WO2021004838A2/en
Publication of WO2021004838A3 publication Critical patent/WO2021004838A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8269—Photosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01039—Ribulose-bisphosphate carboxylase (4.1.1.39)

Definitions

  • the present invention relates to methods and means to increase the ratio of Rubisco Activase (Rca) proteins having higher activity and reduced sensitivity to ADP inhibition in plants and increase the photosynthesis and yield of plants.
  • the invention provides a Rca a protein having an increased protein activity and a reduced sensitivity to ADP inhibition compared to the native Rca a proteins.
  • Ribulose-l,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is the central enzyme of photosyn- thesis catalyzing the fixation of C02 into sugars (Andersson, 2008, J. Exp. Bot. 59, 1555-1568). Rubisco is susceptible to inhibition by catalytic misfire products, in a process sometimes referred to as failover ( Pearce, F. G., and Andrews, T. J., 2003, J. Biol. Chem. 278, 32526-32536; Pearce, F. G., 2006, Biochem. J. 399, 525-534; Schrader et al., 2006, Funct. Plant Biol. 33, 921-929).
  • RuBP Ribulose-l,5-bisphosophate
  • the sugar substrate of Rubisco, Ribulose-l,5-bisphosophate is an inhibitor of Ru- bisco (often referred to as the ER complex) if the active site is not initially primed by the binding of a Mg2+ ion and carbamylated with a molecule of C02 separate from the one catalyzed (often referred to as the ECM complex) (Jordan, D. B., and Chollet, R., 1983, J. Biol. Chem. 258, 13752- 13758; Cleland et al., 1998, Chem. Rev. 98, 549-562).
  • Rca Rubisco activase
  • Rca clears tightly bound sugar substrates and inhibitors from the active site of Rubisco through a mechanism which is not clearly characterized for higher plants (Mueller-Cajar et al., 2014, Photosynth. Res. 119, 191-201). Diffi- culty in characterizing interaction with Rubisco arises as the active multimeric Rca complex is dynamic and altered by co-factors such as nucleotides and Mg2+, as well as concentration and solvent dependent self-association (Henderson et al., 2013, Spec. Sect. Chem. Mech. Phosphatases Diesterases Triesterases. 1834, 87-97; Keown, J. R., and Pearce, F.
  • CTE C-terminal extension
  • a- isoform In some species that express both an a and b-isoform such as Arabidopsis ( Arabidopsis thaliana) the a- isoform is sensitive to ADP inhibition while the b-isoform has relatively minimal sensitivity, while other species such as tobacco ( Nicotiana tabacum) only expresses a b-isoform that is sensitive to ADP (Carmo-Silva, A. E., and Salvucci, M. E., 2013, Plant Physiol. 161, 1645-1655).
  • Rice and wheat express both an a and b-isoform, with wheat expressing an a-isoform (TaRca2-a) and two variants of the b-isoform, the spliced variant of the a, referred to as TaRca2-b and a unique b- isoform encoded by a separate gene, referred to as TaRcal-b ( To et al., 1999, Planta. 209, 66- 76; Carmo-Silva et al., 2015, Plant Cell Environ. 38, 1817-1832). The sensitivity of these wheat isoforms to ADP and ATP is not known.
  • the invention provides a Rca a protein (Rubisco activase) comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
  • the Rca a protein may comprise an amino acid sequence selected from (a) the amino acid sequence of SEQ ID NOs: 3, 7; 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87, wherein the amino acid at a position corresponding to position 382 of SEQ ID NO: 7 is substituted with an arginine (b) an amino acid sequence having at least 90% identity to the amino acid sequences of SEQ ID NOs: 3, 7; 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87 and comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7, (c) the amino acid sequences of SEQ ID NOs:
  • This Rca a protein may have an increased maximal Rubisco activase activity and/or a reduced ADP inhibition of its Rubisco activase activity compared to an Rca 2a protein not comprising said arginine at a position corresponding to position 382 of SEQ ID NO: 7.
  • a nucleic acid encoding the Rca a protein according to the invention is also provided and it may comprise a coding nucleotide sequence selected from (a) the nucleotide sequence of SEQ ID Nos: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88, wherein the codon encoding the amino acid at a position corresponding to position 382 of SEQ ID NO: 7 is substituted with an codon encoding an arginine, or the complement thereof, and (b) a nucleotide sequence having at least 60% identity with the nucleotide sequence of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88 and encoding the Rca a protein according to the invention, or the complement thereof.
  • a gene such as a recombinant gene, which comprises the following operably linked elements (a) a promoter, preferably expressible in plants, (b) a nucleic acid encoding the Rca a protein according to the invention and, optionally (c) a transcription ter- mination and polyadenylation region, preferably a transcription termination and polyadenylation region functional in plants.
  • Said promoter may be a constitutive promoter, tissue-specific promoter or an inducible promoter and said nucleic acid encoding the Rca a protein may comprise the coding nucleotide sequence according to the invention.
  • a cell which comprises the protein of the invention, the nucleic acid of the invention or at least one Rca a allele according to the invention.
  • the plant cell may further comprise (a) at least one knock-out allele of a Rca gene, or (b) a recombinant gene capable of suppressing specifically the expression of the endogenous Rca genes.
  • a plant, plant part or seed consisting essentially of the plant cells of the invention are also provided.
  • a method of increasing the maximal Rubisco activase activity and/or of reducing the ADP inhibition of the Rubisco activase activity of a Rca a protein comprises introducing an amino acid substitution to the amino acid sequence of said Rca a protein, wherein the amino acid substitution is a substitution of the amino acid at a position corresponding to position 382 of SEQ ID NO: 7 with an arginine.
  • the maximal Rubisco activase activity may be increased by about 10% and the ADP inhibition of the Rubisco activase activity may be reduced by about 10%.
  • a method for increasing the ratio of an arginine Rca a protein variant in plants comprising (a) providing to cells of a plant the recombinant gene provided herein, and re- ducing the expression of endogenous Rca protein in said plant cells, wherein said ratio is increased compared to a control plant cell not comprising said recombinant gene; or (b) introducing into cells of a plant at least one Rca a allele according to the invention, wherein said ratio is increased com- pared to a control plant cell not comprising said Rca a allele.
  • endogenous Rca a protein may be reduced by (a) introducing into said cells of a plant at least one knock-out mutant Rca allele; or (b) providing said cells of a plant with a second recombinant gene capable of sup- pressing specifically the expression of the endogenous Rca genes.
  • a method for increasing photosynthetic activity and/or increasing yield of a plant is also pro- vided which comprises (a) increasing the ratio of an arginine Rca a protein variant, and (b) regen- erating said plant, wherein said photosynthetic activity and/or yield is increased compared to a plant not comprising said increased ratio of an arginine Rca a protein variant.
  • the yield may be seed yield or thousand seed weight.
  • a method for producing a plant with an increased photosynthetic activity and /or with an in- creased yield comprises (a) increasing the ratio of an arginine Rca a protein variant according to the invention in a plant and (b) regenerating said plant, wherein said photosynthetic activity and/or said yield is increased compared to a plant not comprising said in- creased ratio of an arginine Rca a protein variant.
  • a method for producing a plant with an increased photosynthetic activity and/or increased yield comprises (a) increasing the ratio of an arginine Rca a protein variant; and (b) regenerating said plant, wherein said photosynthetic activity and/or yield is increased com- pared to a plant not comprising said increased ratio of an arginine Rca a protein variant.
  • arginine Rca a protein variant according to the invention the nucleic acid encoding an arginine Rca a protein variant according to the invention, the recombinant gene according to the invention or the arginine Rca a allele according to the invention, to increase the ratio of an arginine Rca a protein variant in plant, to increase photosynthetic activity of a plant, to increase yield of a plant or to produce a plant with increased photosynthetic activity or with an increased yield.
  • FIG. 1 ADP inhibition of Rca activation velocity for wheat (TaRca) and rice (OsRca) a and b isoforms.
  • Rca velocity in the presence of 5 mM ATP substrate and varying concentrations of ADP inhibitor was normalized to initial velocity in the absence of inhibitor (V i /V 0 ) and plotted against the fraction of ADP to ATP. Values are the means ⁇ SD of 3 experimental replicates.
  • FIG. 1 C-terminal alignment of wheat (TaRca) and Arabidopsis (AtRca) a and b Rca isoforms indicating the mutation made in this study.
  • the amino-acid position 428 for the wheat TaRca2-a isoform and 432 for the wheat TaRca1-b isoform were mutated from a native lysine (K) to either an arginine (R) or glutamine (Q).
  • FIG. 17 ATP substrate dependent enzyme kinetic curves of the TaRca2-a isoform and de- rived CTE mutant (A), and wheat b isoforms and TaRca1-b derived mutant (B).
  • the enzymatic velocity of Rubisco reactivation by Rca (ECM regenerated reactions per minute per Rca monomer) was plotted against ATP concentration added to assays.
  • An ATP regenerating system using phos- phocreatine and creatine phosphokinase was used to limit ADP buildup due to ATP hydrolysis by Rca.
  • V max , K half and h values were generated from iterative fits using a least-squares model and values for each curve are presented in Table 2. Values are the means ⁇ SD of 3-5 experimental replicates.
  • FIG. 4 Inhibitor dose-response curves of TaRca2-a and C-terminal extension mutant (A), and for the wheat b isoforms and TaRca1-b mutant (B). The relationship between IC 50 and K half for the a variants (C) and b variants (D).
  • the present invention is based on the surprising discovery that substituting the amino acid at the position corresponding to position 382 of SEQ ID NO: 7 with an arginine reduces the ADP inhibition of the Rca a protein activity and increases the maximal Rubisco activase ( Rca) activity of the Rca a protein.
  • percent sequence identity refers to the per- centage of conserved amino acids between two segments of a window of optimally aligned poly- peptides.
  • Optimal alignment of sequences for aligning a comparison window are well-known to those skilled in the art and the percentage of conservation may be calculated by matrix such as BLOSUM (Blocks Substitution Matrix) and PAM (Point Accepted Mutation) (Henikoff and Heni- koff, 1992, PNAS 89(22): 10915-10919).
  • identity fraction for aligned segments of a test se- quence and a reference sequence is the number of identical or conserved components that are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction times 100.
  • the comparison of one or more protein sequences may be to a full-length protein sequence or a portion thereof, or to a longer protein sequence.
  • protein interchangeably used with the term“polypeptide” as used herein describes a group of molecules consisting of more than 30 amino acids, whereas the term“peptide” describes molecules consisting of up to 30 amino acids. Proteins and peptides may further form dimers, tri- mers and higher oligomers, i.e. consisting of more than one (poly)peptide molecule. Protein or peptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corre- sponding higher order structures are, consequently, termed homo- or heterodimers, homo- or het- erotrimers etc.
  • protein and“peptide” also refer to naturally modified proteins or pep- tides wherein the modification is effected e.g. by glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.
  • variant nucleotide sequences of the invention is intended to mean substantially similar sequences.
  • Naturally occurring allelic variants such as these can be iden- tified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as herein outlined before.
  • variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis of any one of SEQ ID NOs: 1, 5; 9, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 or 85.
  • nucleotide sequence variants of the invention will have at least 40%, at least 50%, at least 60%, to at least 70%, e.g., preferably at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, to at least 79%, generally at least 80%, e.g., at least 81% to at least 84%, at least at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, to at least 98% and at least 99% nucle- otide sequence identity to the native (wild type or endogenous) nucleotide sequence.
  • Derivatives of the DNA molecules disclosed herein may include, but are not limited to, deletions of sequence, single or multiple point mutations, alterations at a particular restriction enzyme site, addition of functional elements, or other means of molecular modification. Techniques for obtaining such de- rivatives are well-known in the art (see, for example, J. F. Sambrook, D. W. Russell, and N. Irwin (2000) Molecular Cloning: A Laboratory Manual, 3 rd edition Volumes 1, 2, and 3. Cold Spring Harbor Laboratory Press).
  • the term "percent sequence identity" refers to the percentage of identical nucleotides between two segments of a window of optimally aligned DNA. Optimal alignment of sequences for aligning a comparison window are well-known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Wa- terman (Waterman, M. S. Introduction to Computational Biology: Maps, sequences and genomes.
  • identity fraction for aligned segments of a test sequence and a reference sequence is the num- ber of identical components that are shared by the two aligned sequences divided by the total num- ber of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the iden- tity fraction times 100.
  • the comparison of one or more DNA sequences may be to a full-length DNA sequence or a portion thereof, or to a longer DNA sequence.
  • recombinant gene refers to any artificial gene that contains: a) DNA sequences, including regulatory and coding sequences that are not found together in nature, or b) sequences encoding parts of proteins not naturally adjoined, or c) parts of promoters that are not naturally adjoined. Accordingly, a recombinant gene may comprise regulatory sequences and coding se- quences that are derived from different sources, i.e. heterologous sequences, or comprise regulatory sequences, and coding sequences derived from the same source, but arranged in a manner different from that found in nature.
  • heterologous refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources.
  • a promoter is heterologous with respect to an operably linked DNA region, such as a coding sequence if such a combination is not normally found in nature.
  • a particular sequence may be "heterologous” with respect to a cell or organism into which it is inserted (i.e. does not naturally occur in that particular cell or organism).
  • the recombinant gene disclosed herein is a heterologous nucleic acid.
  • endogenous relates to what originate from within the plant or cell.
  • An endogenous gene is thus a gene originally found in a given plant or cell.
  • Isolated nucleic acid used interchangeably with“isolated DNA” as used herein refers to a nucleic acid not occurring in its natural genomic context, irrespective of its length and sequence.
  • Isolated DNA can, for example, refer to DNA which is physically separated from the genomic context, such as a fragment of genomic DNA.
  • Isolated DNA can also be an artificially produced DNA, such as a chemically synthesized DNA, or such as DNA produced via amplification reac- tions, such as polymerase chain reaction (PCR) well-known in the art.
  • Isolated DNA can further refer to DNA present in a context of DNA in which it does not occur naturally. For example, iso- lated DNA can refer to a piece of DNA present in a plasmid.
  • the isolated DNA can refer to a piece of DNA present in another chromosomal context than the context in which it occurs naturally, such as for example at another position in the genome than the natural position, in the genome of another species than the species in which it occurs naturally, or in an artificial chromo- some.
  • Hybridization occurs when the two nucleic acid molecules anneal to one another under appro- priate conditions.
  • Nucleic acid hybridization is a technique well known to those of skill in the art of DNA manipulation.
  • the hybridization property of a given pair of nucleic acids is an indication of their similarity or identity.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.
  • the phrase "hybridizing specifically to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a com- plex mixture (e.g., total cellular) DNA or RNA.
  • “Bind(s) substantially” refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mis- matches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
  • “Stringent hybridization condi- tions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence-dependent, and are differ- ent under different environmental parameters.
  • An example of highly stringent wash conditions is 0.15 M NaCI at 72°C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2 X SSC wash at 65°C for 15 minutes.
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a signal to noise ratio of 2 X (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This oc- curs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • the phrases“DNA”, “DNA sequence,” “nucleic acid sequence,” “nucleic acid molecule”“nu- cleotide sequence” and“nucleic acid” refer to a physical structure comprising an orderly arrange- ment of nucleotides.
  • the terms“sequence” and“molecule” may be used interchangeably.
  • the DNA sequence or nucleotide sequence may be contained within a larger nucleotide molecule, vector, or the like.
  • the orderly arrangement of nucleic acids in these sequences may be depicted in the form of a sequence listing, figure, table, electronic medium, or the like.
  • nucleic acid or protein comprising a sequence of nucleotides or amino acids
  • a recombinant gene comprising a nucleic acid which is functionally or structurally defined may comprise additional DNA regions etc.
  • the term “comprising” also includes“consisting of’.
  • allele(s) means any of one or more alternative forms of a gene at a particular locus.
  • alleles of a given gene are located at a specific location or locus (loci plural) on a chromosome.
  • loci plural locus
  • One allele is present on each chromosome of the pair of homologous chromosomes.
  • the term“locus” (loci plural) means a specific place or places or a site on a chromosome where for example a gene or genetic marker is found.
  • the“ TaRca 2 A locus” refers to the position on a chromosome of the wheat A genome where a TaRca 2 A gene (and two TaRca 2 A alleles) may be found
  • the“TaRca 2 B locus” refers to the position on a chromosome of the wheat B genome where a TaRca 2 B gene (and two TaRca 2B alleles) may be found
  • the“TaRca 2 D locus” refers to the position on a chromosome of the wheat D genome where a TaRca 2 D gene (and two TaRca 2 D alleles) may be found.
  • the“ OsRca locus” refers to the position on a chromosome of the rice genome where an Os Rca gene (and two Os Rca alleles) may be found
  • the“ BnRca 1 A locus” refers to the position on a chromosome of the Brassica napus A genome where a BnRca 1 A gene (and two BnRca 1 A alleles) may be found
  • the“ BnRca 2 A locus” refers to the position on a chromosome of the Brassica napus A genome where a BnRca 2 A gene (and two BnRca 2 A alleles) may be found
  • the“ BnRca 3 A locus” refers to the position on a chromosome of the Brassica napus A genome where a BnRca 3 A gene (and two BnRca 3 A alleles) may be found
  • the“ BnRca 1 C locus” refers to the position on a chromosome of the Brassica napus
  • An Rca gene as used herein is a gene from which an iso aform of an Rca protein can be pro- prised.
  • the same Rca gene may or may not also produce a b isoform of an Rca protein.
  • Wild type refers to a typical form of a plant or a gene as it most commonly occurs in nature.
  • A“wild type plant” refers to a plant with the most common phenotype of such plant in the natural population.
  • A“wild type allele” refers to an allele of a gene required to produce the wild-type protein and wild type phenotype.
  • a“mutant plant” refers to a plant with a different rare phenotype of such plant in the natural popu- lation or produced by human intervention, e.g. by mutagenesis or gene editing, and a“mutant al- lele” refers to an allele of a gene required to produce the mutant protein and/or the mutant pheno- type.
  • “Mutant” as used herein refers to a form of a plant or a gene which is different from such plant or gene in the natural population, and which is produced by human intervention, e.g. by mutagen- esis or gene editing
  • a“mutant allele” refers to an allele which is not found in plants in the natural population or breeding population, but which is produced by human intervention such as mutagenesis or gene editing.
  • the term“wild type allele” e.g. wild type TaRca 2 B allele, wild type TaRca
  • wild type TaRca 2 D allele wild type OsRca allele, wild type BnRca 1 A allele, wild type BnRca 2 A allele, wild type BnRca 3 A allele, wild type BnRca 1 C allele, wild type BnRca 2 C allele, wild type BnRca 3 C allele, wild type GhRca 1 A allele, wild type GhRca 2 A allele, wild type GhRca 1 D allele, wild type GhRca 2 D allele, wild type GmRca 1 allele, wild type GmRca 2 allele, wild type GmRca 3 allele, wild type ZmRca allele, or a wild type AtRca allele), means a naturally occurring allele found in plants, in particular wheat plants, rice plants, Brassica plants, soybean plants, cotton plants, maize plants or Arabidopsis plants, which encodes a functional a isoform Rca protein (e.g.
  • an Rca allele encoding a non-functional iso aform Rca protein e.g. a non-functional isofo arm TaRca 2 B, i asoform TaRca 2 A, a isoform TaRca 2 D, iso aform OsRca, isof aorm BnRca 1 A, a isoform BnRca 2 A, is aoform BnRca 3 A, a isoform BnRca 1 C, a isoform BnRca 2 C, isoform a BnRca 3 C, a isoform GhRca 1 A, a isoform GhRca 2 A, iso aform GhRca 1 D, a isoform GhRca 2 D, a isoform GmRca 1, is aoform GmRca 2, iso aform GmRca 3, isofo arm ZmRca, or isoform a AtRca protein) or an Rca allele encoding
  • a functional i asoform TaRca 2 B is aoform TaRca 2 A, iso aform TaRca 2 D, isof aorm OsRca, a isoform BnRca 1 A, a isoform BnRca 2 A, isof aorm BnRca 3 A, a isoform BnRca 1 C, a isoform BnRca 2 C, a isoform BnRca 3 C, a isoform GhRca 1 A, a isoform GhRca 2 A, isof aorm GhRca 1 D, a isoform GhRca 2 D, a isoform GmRca 1, isofo arm GmRca 2, isofor am GmRca 3, isoform a ZmRca, or a isoform AtRca protein having an increase maximal Rca activity and/or a reduced ADP inhibition of its Rca activity compared to a wild type iso aform protein).
  • a mutant Rca allele en- coding a non-functional a isoform Rca protein refers to an iso aform Rca protein having no biological activity or a significantly reduced biological activity as compared to the cor- responding wild-type functional i asoform Rca protein, or encoding no is aoform Rca protein at all.
  • a knock-out Rca allele is an equivalent term for a mutant Rca allele encoding a non-functional isof aorm Rca protein.
  • A“382R” Rca allele is an equivalent term for an Rca allele encoding a functional isof aorm protein having an increase maximal Rca activity and/or a reduced ADP inhibition of its Rca activity compared to a wild type allele.
  • “Mutagenesis”, as used herein, refers to the process in which plant cells (e.g., a plurality of cereal seeds or other parts, such as pollen, etc.) are subjected to a technique which induces muta- tions in the DNA of the cells, such as contact with a mutagenic agent, such as a chemical substance (such as ethylmethylsulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation (neutrons (such as in fast neutron mutagenesis, etc.), alpha rays, gamma rays (such as that supplied by a Cobalt 60 source), X-rays, UV-radiation, etc.), T-DNA insertion mutagenesis (Azpiroz-Leehan et al.
  • a mutagenic agent such as a chemical substance (such as ethylmethylsulfonate (EMS), ethylnitrosourea
  • the desired mutagenesis of one or more Rca 2 alleles may be accomplished by use of one of the above methods. While mutations created by irradiation are often large deletions or other gross lesions such as translocations or complex rearrangements, mutations created by chemical mutagens are often more discrete lesions such as point mutations.
  • EMS alkylates guanine bases which results in base mispairing: an alkylated guanine will pair with a thymine base, resulting primarily in G/C to A/T transitions.
  • cereal plants are regenerated from the treated cells using known techniques. For instance, the resulting cereal seeds may be planted in accordance with conventional growing procedures and following self- pollination seed is formed on the plants. Additional seed that is formed as a result of such self- pollination in the present or a subsequent generation may be harvested and screened for the pres- ence of mutant Rca alleles.
  • DeleteageneTM Delete -a-gene; Li et al., 2001, Plant J 27: 235-242
  • PCR polymerase chain reaction
  • Gene editing refers to the targeted modification of genomic DNA using se- quence-specific enzymes (such as endonuclease, nickases, base conversion enzymes) and/or donor nucleic acids (e.g. dsDNA, oligo’s) to introduce desired changes in the DNA.
  • se- quence-specific enzymes such as endonuclease, nickases, base conversion enzymes
  • donor nucleic acids e.g. dsDNA, oligo’s
  • Sequence-specific nucleases that can be programmed to recognize specific DNA sequences include meganucleases (MGNs), zinc-finger nucleases (ZFNs), TAL-effector nucleases (TALENs) and RNA-guided or DNA-guided nucleases such as Cas9, Cpfl, CasX, CasY, C2cl, C2c3, certain Argonaut-based sys- tems (see e.g. Osakabe and Osakabe, Plant Cell Physiol. 2015 Mar; 56(3):389-400; Ma et al., Mol Plant.
  • MGNs meganucleases
  • ZFNs zinc-finger nucleases
  • TALENs TAL-effector nucleases
  • RNA-guided or DNA-guided nucleases such as Cas9, Cpfl, CasX, CasY, C2cl, C2c3, certain Argonaut-based sys- tems (see
  • Donor nucleic acids can be used as a template for repair of the DNA break induced by a sequence specific nuclease but can also be used as such for gene targeting (without DNA break induction) to introduce a de- sired change into the genomic DNA. Sequence-specific nucleases may also be used without donor nucleic acid, thereby allowing insertion or deletion mutations via non-homologous end joining re- pair mechanism. Gene editing can be used to create mutant Rca alleles.
  • Mutant nucleic acid molecules or mutant alleles may comprise one or more mutations or mod- ifications, such as: a. a“missense mutation”, which is a change in the nucleic acid sequence that results in the substitution of an amino acid for another amino acid;
  • a“nonsense mutation” or“STOP codon mutation” which is a change in the nucleic acid sequence that results in the introduction of a premature STOP codon and thus the termina- tion of translation (resulting in a truncated protein); plant genes contain the translation stop codons“TGA” (UGA in RNA),“TAA” (UAA in RNA) and“TAG” (UAG in RNA); thus any nucleotide substitution, insertion, deletion which results in one of these codons to be in the mature mRNA being translated (in the reading frame) will terminate translation; c. an“insertion mutation” of one or more amino acids, due to one or more codons having been added in the coding sequence of the nucleic acid;
  • a frameshift mutation can have various causes, such as the insertion, deletion or duplication of one or more nucleotides; f. a mutated splice site, resulting in altered splicing, which results in an altered mRNA pro- cessing and, consequently, in an altered encoded protein which contains either deletions, substitutions or insertions of various lengths, possibly combined with premature translation termination.
  • Rca proteins and nucleic acids can have various causes, such as the insertion, deletion or duplication of one or more nucleotides; f. a mutated splice site, resulting in altered splicing, which results in an altered mRNA pro- cessing and, consequently, in an altered encoded protein which contains either deletions, substitutions or insertions of various lengths, possibly combined with premature translation termination.
  • Rca proteins are AAA+ chaperones which can form hexameric protein complexes and interact with Rubisco.
  • Functional Rca proteins comprise a central ATPase domain (the AAA+ module) and a C-terminal domain involved in Rubisco-Rca and Rca-Rca interactions 33-35 .
  • the AAA+ module is located from amino acid at a position equivalent to position 57 to a position equivalent to position 345 on SEQ ID NO: 5
  • the ATPase core is located from amino acid at a position equivalent to position 182 to a position equivalent to position 282 on SEQ ID NO: 5
  • the C-terminal domain is located from amino acid at a position equivalent to position 346 to a position equivalent to posi- tion 427 on SEQ ID NO: 5.
  • functional Rca proteins furthermore comprise an N- linker (IA) at the amino acid positions equivalent to position 123 and 124 on SEQ ID NO: 5, a Walker A motif (GxxxxGK) at the amino acid positions equivalent to positions 155 to 161 on SEQ ID NO: 5, a Walker B motif (LxxxD) at the amino acid positions equivalent to positions 215 to 219 on SEQ ID NO: 5, a Rubisco interaction loop (Shivhare et al 2017) at the amino acid positions equivalent to positions 253 to 265 on SEQ ID NO: 5, an Rca-Rca interface (Stotz et al.
  • IA N- linker
  • GxxxxGK Walker A motif
  • LxxxD Walker B motif
  • Complexes comprising the Rubisco Activase and the Rubisco protein may be formed in vitro or in vivo.
  • the complexes may be formed in vitro by contacting a 382R Rca protein according to the invention with Rubisco from wheat, rice, Brassica, cotton, maize, soybean, Ara- bidopsis, or other plant species present in a leaf extract or Rubisco that has been purified from wheat, rice, Brassica, cotton, maize, soybean, Arabidopsis, or other plant species.
  • the complexes may be formed in vivo by expression of an Rca polypeptide of the invention in a plant such that it forms a complex with the endogenous Rubisco of the plant.
  • the endogenous Rca a protein may comprise the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 or 87.
  • the endogenous Rca a protein may also comprise an amino acid sequence having at least 90% sequence identity with the amino acid se- quences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 or 87 and not comprise an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
  • An endogenous Rca a protein may comprise an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% sequence identity to the above described protein which does not comprise an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
  • SEQ ID NOs: 1 and 3 represent the amino acid sequences of the a isoform of the TaRca 2 A protein from wheat including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 5 and 7 represent the amino acid sequences of the TaRca 2a protein from the wheat sub- genome B including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 9 and 11 represent the amino acid sequences of the TaRca 2a protein from the wheat subgenome D including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 25 and 27 represent the amino acid sequences of the rice OsRca p arotein including and excluding, respec- tively, the chloroplast targeting peptide.
  • SEQ ID NOs: 29 and 31 represent the amino acid se- quences of the BnRca 1a protein from the Brassica napus subgenome A including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 33 and 35 represent the amino acid sequences of the BnRca 2a protein from the Brassica napus subgenome A including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 37 and 39 represent the amino acid sequences of the BnRca 3a protein from the Brassica napus subgenome A including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 41 and 43 represent the amino acid sequences of the BnRca 1a protein from the Brassica napus subgenome C including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 45 and 47 represent the amino acid sequences of the BnRca 2a protein from the Brassica napus subgenome C including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 49 and 51 represent the amino acid sequences of the BnRca 3a protein from the Brassica napus subgenome C including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 53 and 55 represent the amino acid sequences of the GhRca 1a protein from the Gossypium hirsutum subgenome A including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 57 and 59 represent the amino acid sequences of the GhRca 2a protein from the Gossypium hirsutum subgenome A includ- ing and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 61 and 63 represent the amino acid sequences of the GhRca la protein from the Gossypium hirsutum subgenome D including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 65 and 67 represent the amino acid sequences of the GhRca 2a protein from the Gossypium hirsutum subge- nome D including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 69 and 71 represent the amino acid sequences of the soybean GmRca la protein including and exclud- ing, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 73 and 75 represent the amino acid sequences of the soybean GmRca 2a protein including and excluding, respectively, the chlo- roplast targeting peptide.
  • SEQ ID NOs: 77 and 79 represent the amino acid sequences of the soy- bean GmRca 3a protein including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 81 and 83 represent the amino acid sequences of the maize ZmRca prote ain includ- ing and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 85 and 87 represent the amino acid sequences of the Arabidopsis AtRca pro atein including and excluding, respectively, the chloroplast targeting peptide.
  • the endogenous Rca genes encoding said endogenous Rca a proteins may com- prise the coding nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86 or 88.
  • SEQ ID NOs: 2 and 4 represent the coding nucleotide sequences of the a isoform of the TaRca 2 A gene from wheat including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 6 and 8 represent the coding nucleotide sequences of the TaRca 2a gene from the wheat subgenome B including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 10 and 12 represent the coding nucleotide sequences of the TaRca 2a gene from the wheat subgenome D including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 26 and 28 represent the coding nucleotide sequences of the rice OsRca gene a including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 30 and 32 represent the coding nucleotide sequences of the BnRca 1 gen ae from the Brassica napus subgenome A including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 34 and 36 represent the coding nucleotide sequences of the BnRca 2a gene from the Brassica napus subgenome A in- cluding and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 38 and 40 represent the coding nucleotide sequences of the BnRca 3a gene from the Brassica napus subge- nome A including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 42 and 44 represent the coding nucleotide sequences of the BnRca la gene from the Brassica napus subgenome C including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 46 and 48 represent the coding nucleotide sequences of the BnRca 2a gene from the Brassica napus subgenome C including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 50 and 52 represent the coding nucleotide sequences of the BnRca 3a gene from the Brassica napus subgenome C including and excluding, respectively, the chloroplast targeting pep- tide.
  • SEQ ID NOs: 54 and 56 represent the coding nucleotide sequences of the GhRca la gene from the Gossypium hirsutum subgenome A including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 58 and 60 represent the coding nucleotide sequences of the GhRca 2a gene from the Gossypium hirsutum subgenome A including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 62 and 64 represent the coding nucleotide sequences of the GhRca la gene from the Gossypium hirsutum subgenome D including and excluding, re- spectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 66 and 68 represent the coding nucleo- tide sequences of the GhRca 2a gene from the Gossypium hirsutum subgenome D including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 70 and 72 represent the coding nucleotide sequences of the soybean GmRca la gene including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 74 and 76 represent the coding nucleotide se- quences of the soybean GmRca 2a gene including and excluding, respectively, the chloroplast tar- geting peptide.
  • SEQ ID NOs: 78 and 80 represent the coding nucleotide sequences of the soybean GmRca 3a gene including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 82 and 84 represent the coding nucleotide sequences of the maize ZmRca gen ae including and excluding, respectively, the chloroplast targeting peptide.
  • SEQ ID NOs: 86 and 88 represent the coding nucleotide sequences of the Arabidopsis AtRca gen ae including and excluding, respec- tively, the chloroplast targeting peptide.
  • the endogenous Rca a gene encoding said endogenous Rca a gene may also comprise a coding nucleotide sequence having at least 60% identity with the nucleotide sequence of SEQ ID NOs: 2,
  • a nucleic acid comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86 or 88 and not encoding an arginine at a position corresponding to position 382 of SEQ ID NO: 7 may be a nucleic acid comprising a nucleotide sequence having at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or 100% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70
  • a knock-out allele of an Rca a gene is disclosed herein.
  • the knock-out allele of an Rca a gene may be a full knock-out allele or a partial knock-out allele.
  • A“full knock-out” or“null” allele refers to a mutant allele, which encodes a protein having no biological activity as compared to the corresponding wild-type functional protein or which encodes no protein at all.
  • Such a“full knock-out mutant allele” is, for example, a wild- type allele, which comprises one or more mutations in its nucleic acid sequence, for example, one or more non-sense, mis-sense, insertion, deletion, frameshift or mutated splice site mutations.
  • such a full knock-out mutant Rca a allele is a wild-type Rca a allele, which comprises a mutation that preferably result in the production of an Rca a protein lacking at least one functional domain or motif, such as the central ATPase domain (the AAA+ module), the C-terminal domain, the N-linker, Walker A, Walker B motives, the Rubisco interaction loop, the Rca-Rca interface, or lacking at least one amino acid critical for its function such as the tyrosine (Y) at the amino acid positions equivalent to position 406 of SEQ ID NO: 5, such that the biological activity of the Rca a protein is completely abolished, or whereby the modification(s) preferably result in no production of an Rca a protein.
  • the central ATPase domain the AAA+ module
  • the C-terminal domain the N-linker
  • Walker A Walker B motives
  • the Rubisco interaction loop the Rca-Rca interface
  • at least one amino acid critical for its function such as the tyros
  • A“partial knock-out” mutant allele refers to a mutant allele, which encodes a protein having a significantly reduced biological activity as compared to the corresponding wild- type functional protein.
  • Such a“partial knock-out mutant allele” is, for example, a wild-type allele, which comprises one or more mutations in its nucleic acid sequence, for example, one or more missense mutations.
  • such a partial knockout mutant allele is a wild-type allele, which comprises a mutation that preferably results in the production of a protein wherein at least one conserved and/or functional amino acid is substituted for another amino acid, such that the biolog- ical activity is significantly reduced but not completely abolished.
  • a missense mutation in an Rca a allele is any mutation (deletion, insertion or substitution) in an Rca a allele whereby one or more codons are changed into the coding DNA and the corresponding mRNA sequence of the corresponding wild type Rca a allele , resulting in the substitution of one or more amino acids in the wild type Rca a protein for one or more other amino acids in the mutant Rca a protein.
  • a mutant Rca a allele comprising a missense mutation is an Rca a allele wherein one amino acid is substituted.
  • a nonsense mutation in an Rca all aele is a mutation in an Rca allele w ahereby one or more translation stop codons are introduced into the coding DNA and the corresponding mRNA sequence of the corresponding wild type Rca a allele .
  • Translation stop codons are TGA (UGA in the mRNA), TAA (UAA) and TAG (UAG).
  • the mutant Rca a protein maintains the amino acids encoded by the coding DNA upstream of the mutation (i.e. the N-terminal part of the Rca a protein).
  • Table 1 Examples of substitution mutation resulting in the generation of an in-frame stop codon.
  • a frameshift mutation in an Rca a allele is a mutation (deletion, insertion, duplication, and the like) in an Rca all aele that results in the nucleic acid sequence being translated in a different frame downstream of the mutation.
  • a splice site mutation in an Rca a allele is a mutation (deletion, insertion, substitution, duplication, and the like) in an Rca a allele whereby a splice donor site or a splice acceptor site is mutated, resulting in altered processing of the mRNA and, consequently, an altered encoded protein, which can have insertions, deletions, substitutions of various lengths, or which can be truncated.
  • a deletion mutation in an Rca a allele is a mutation in an Rca a allele that results in the production of an Rca a protein which lacks the amino acids encoded by the deleted coding DNA and maintains the amino acids encoded by the coding DNA upstream of the deletion (i.e. the N-terminal part of the Rca a protein) and encoding by the coding DNA downstream of the deletion (i.e. the C-terminal part of the Rca a protein).
  • A“significantly reduced amount of functional Rca a protein” refers to a reduction in the amount of a functional protein produced by the cell comprising a mutant Rca alle ale by at least 30%, 40%, 50%, 60%, 70%, or 80% as compared to the amount of the functional Rca a protein produced by the cell not comprising the mutant Rca a allele. The production of functional Rca a protein is however not abolished.
  • This definition encompasses the production of a“non-functional” Rca a protein (e.g. truncated Rca a protein) having reduced biological activity in vivo, the reduction in the absolute amount of the functional Rca a protein (e.g. no functional Rca a protein being made due to the mutation in the Rca a gene), the production of an Rca a protein with significantly reduced biological activity compared to the activity of a functional wild type Rca a protein (such as an Rca a protein in which one or more amino acid residues that are crucial for the biological activity of the encoded Rca a protein are substituted for another amino acid residue).
  • a“non-functional” Rca a protein e.g. truncated Rca a protein having reduced biological activity in vivo
  • the reduction in the absolute amount of the functional Rca a protein e.g. no functional Rca a protein being made due to the mutation in the Rca a gene
  • an“arginine Rca a protein variant” is provided.
  • Such arginine Rca a protein variant is an Rca a protein wherein the amino acid at position corresponding to position 382 on SEQ ID NO: 7 is replaced with, or substituted with, an arginine.
  • the arginine Rca a variant may comprise an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 3, 7, 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87, where the amino acid at position equivalent to position 382 of SEQ ID NO: 7 is substituted with an arginine or an amino acid sequence having at least 90% identity to the amino acid sequences of SEQ ID NOs: 3, 7, 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87 and comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
  • the arginine Rca a protein variant may also comprise an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 3, 7, 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87, where the amino acid at position equivalent to position 382 of SEQ ID NO: 7 is substituted with an arginine, and further comprising a chloroplast targeting peptide; or an amino acid sequence having at least 90% identity to the amino acid sequences of SEQ ID NOs: 3, 7, 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87, further comprising a chloroplast targeting peptide and comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
  • the arginine Rca a variant may comprise an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1, 5, 9, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 or 85, where the amino acid at position equivalent to position 382 of SEQ ID NO: 7 is substituted with an arginine; or an amino acid sequence having at least 90% identity to the amino acid se- quences of SEQ ID NOs: 1, 5, 9, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 or 85 and comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
  • the arginine Rca a protein variant may comprise an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% sequence identity to the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 or 87 and comprise an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
  • arginine variants of Rca a proteins wherein one or more amino acid residues have been deleted, substituted or inserted, can also be used to the same effect in the meth- ods according to the invention, provided that the central ATPase domain (the AAA+ module), the C-terminal domain, the N-linker, Walker A, Walker B motives, the Rubisco interaction loop, the Rca-Rca interface and the tyrosine (Y) at the amino acid positions equivalent to position 406 of SEQ ID NO: 5 are not affected by the deletion, substitution or insertion of amino-acid.
  • the central ATPase domain the AAA+ module
  • the C-terminal domain the N-linker
  • Walker A Walker B motives
  • the Rubisco interaction loop the Rca-Rca interface
  • Y tyrosine
  • chloroplast targeting peptides which enable the subcellular tar- geting of the Rca proteins according to the invention to the chloroplast.
  • Chloroplast transit peptide, chloroplast targeting sequence and stromal -targeting transit peptide are equivalent terms.
  • Chloro- plast targeting peptides are recognizable based on the presence of three domains: an uncharged N- terminal domain of about 10 residues beginning with a methionine followed by an alanine and terminating with a glycine or a proline, a central domain lacking acidic residues but enriched in serines and threonines and a C-terminal domain enriched in arginines and forming an amphiphilic b strand (Bruce, 2000, trends in cell biology, Vol 10, 440-447).
  • Such chloroplast targeting peptides are identified herein as the amino acid sequence from po- sition 1 to position 46 of SEQ ID NOs: 1, 9 or 25, the amino acid sequence from position 1 to position 48 of SEQ ID NO: 5, the amino acid sequence from position 1 to position 47 of SEQ ID NOs: 13, 17 or 21, the amino acid sequence from position 1 to position 59 of SEQ ID NO: 29 or 41, the amino acid sequence from position 1 to position 58 of SEQ ID NO: 33 or 45, the amino acid sequence from position 1 to position 52 of SEQ ID NO: 37 or 49, the amino acid sequence from position 1 to position 55 of SEQ ID NO: 53 or 61, the amino acid sequence from position 1 to position 51 of SEQ ID NO: 57 or 65, the amino acid sequence from position 1 to position 60 of SEQ ID NO: 69 or 77, the amino acid sequence from position 1 to position 57 of SEQ ID NO: 73, the amino acid sequence from position 1 to position 35 of SEQ ID NO: 81, the amino acid sequence from position 1 to position 1 to position
  • chloroplast targeting peptides having an amino acid se- quence having at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% identity to the amino acid sequence from position 1 to position 46 of SEQ ID NOs: 1, 9 or 25, the amino acid sequence from position 1 to position 48 of SEQ ID NO: 5, the amino acid sequence from position 1 to position 47 of SEQ ID NOs: 13, 17 or 21, the amino acid sequence from position 1 to position 59 of SEQ ID NO: 29 or 41, the amino acid sequence from position 1 to position 58 of SEQ ID NO: 33 or 45, the amino acid sequence from position 1 to position 52 of SEQ ID NO: 37 or 49, the amino acid sequence from position 1 to position 55 of SEQ ID NO: 53 or 61, the amino acid sequence from position 1 to position 51 of SEQ ID NO: 57 or 65, the amino acid sequence from position 1 to position 60 of SEQ ID NO: 69 or 77, the amino acid sequence from
  • nucleic acid encoding an arginine Rca a protein variant is provided.
  • the nucleic acid encoding an arginine Rca a protein variant may comprise.
  • a coding nucleotide sequence selected from (a) the coding nucleotide sequences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88, where the nucleotides encoding the amino acid at position equivalent to position 382 of SEQ ID NO: 7 are substituted with nucleotides encoding an arginine, or the complement thereof; (b) a coding nucleotide sequence having at least 60% identity to the coding nucleotide sequences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88 and encoding an arginine at position equivalent to position 382 of SEQ ID NO: 7; (c) the coding nucleotide sequences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52,
  • nucleic acids encoding an arginine Rca a protein variant which comprise a nucleotide sequence having at least 40%, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to the herein described gene and are also referred to as variants.
  • a nucleic acid comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88 and encoding an arginine at position equivalent to position 382 of SEQ ID NO: 7 can thus be a nucleic acid com- prising a nucleotide sequence having at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or 100% sequence identity to SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88 and encoding an arginine at position equivalent to position 382 of SEQ ID NO: 7.
  • nucleic acid encoding a chloroplast targeting peptide having the coding nucleotide sequence from position 1 to position 138 of SEQ ID NOs: 2, 10 or 26, the coding nucleotide sequence from position 1 to position 144 of SEQ ID NO: 6, the coding nucleotide sequence from position 1 to position 141 of SEQ ID NOs: 14, 18 or 22, the coding nucleotide sequence from position 1 to position 177 of SEQ ID NO: 30 or 42, the coding nucleotide sequence from position 1 to position 174 of SEQ ID NO: 34 or 46, the coding nucleotide sequence from position 1 to position 156 of SEQ ID NO: 38 or 50, the coding nucleotide sequence from position 1 to position 165 of SEQ ID NO: 54 or 62, the coding nucleotide sequence from position 1 to position 153 of SEQ ID NO: 58 or 66, the coding nucleotide sequence from position 1 to position 180 of SEQ ID
  • nucleic acid encoding a chloroplast targeting peptide having a coding nucleotide sequence having at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or 100% sequence identity to the coding nucleotide sequence from position 1 to position 138 of SEQ ID NOs: 2, 10 or 26, the coding nucleotide se- quence from position 1 to position 144 of SEQ ID NO: 6, the coding nucleotide sequence from position 1 to position 141 of SEQ ID NOs: 14, 18 or 22, the coding nucleotide sequence from position 1 to position 177 of SEQ ID NO: 30 or 42, the coding nucleotide sequence from position 1 to position 174 of SEQ ID NO: 34 or 46, the coding nucleotide sequence from position 1 to position 156 of SEQ ID NO: 38 or 50, the coding nucleotide sequence from position 1 to position 165 of
  • the Arginine Rca a protein variant may be encoded by an“arginine allele” of an Rca a gene.
  • said arginine allele may comprise (a) the coding nucleotide se- quences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88, where the nucleotides encoding the amino acid at position equivalent to position 382 of SEQ ID NO: 7 are substituted with nucleotides encoding an arginine, or the complement thereof; (b) a cod- ing nucleotide sequence having at least 60% identity to the coding nucleotide sequences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88 and encoding an arginine at position equivalent to position 382 of SEQ ID NO: 7; (c) the coding nucleotide se- quences of SEQ ID NOs: 4, 8, 12, 28,
  • endogenous alleles in a plant encoding a Rca a protein can be converted to an arginine Rca a allele by making the desired changes (missense mutations) to existing Rca g aenes, or by replacing one or more endogenous sequences encoding Rca a pro- teins with sequences encoding arginine Rca a protein variants, e.g. as described herein (deletion and insertion mutations).
  • An endogenous allele in a plant such as a cereal plant, wheat, com, Brassica, rice, cotton or soybean, encoding a Rca a protein can also be converted to an arginine Rca a allele by making the desired changes (missense mutations) to existing Rca ge anes using mutagenesis.
  • a recombinant gene comprising the following operably linked elements (a) a promoter, preferably expressible in plants, (b) a nucleic acid encoding a Ar- ginine Rca a protein and, optionally (c) a transcription termination and polyadenylation region, preferably a transcription termination and polyadenylation region functional in plants.
  • the Arginine Rca a protein and nucleic acid variants comprise an amino acid se- quences and nucleotide sequences according to the invention.
  • said pro- moter is a constitutive promoter, tissue-specific promoter or an inducible promoter.
  • the promoter may be a green tissue specific promoter, a mesophyll specific promoter, a light-induced promoter or a temperature induced promoter.
  • a recombinant gene capable of suppressing specifically the expression of the endogenous Rca a genes which comprises the following operably linked elements (a) a promoter, preferably expressible in plants, (b) a nucleic acid which when transcribed yields an RNA molecule inhibitory to the endogenous Rca a genes encoding a Rca a protein not comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7 but not inhibitory to genes encoding Arginine Rca a protein variants; and, optionally (c) a transcription termination and poly- adenylation region, preferably a transcription termination and polyadenylation region functional in plants.
  • the endogenous Rca a genes may comprise the coding nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86 or 88 or a coding nucleotide sequence having at least 60% identity with the nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86 or 88 and not encoding an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
  • Such inhibitory RNA molecule can reduce the expression of a gene for example through the mechanism of RNA-mediated gene silencing. It can be a silencing RNA downregulating expression of a target gene.
  • “silencing RNA” or“silencing RNA molecule” refers to any RNA molecule, which upon introduction into a plant cell, reduces the expression of a target gene.
  • silencing RNA may e.g. be so-called“antisense RNA”, whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the complement of the sequence of the target nucleic acid, preferably the coding sequence of the target gene.
  • antisense RNA may also be directed to regulatory sequences of target genes, including the promoter sequences and transcription termination and polyadenylation signals.
  • Silencing RNA fur- ther includes so-called“sense RNA” whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the sequence of the target nucleic acid.
  • Other silencing RNA may be“unpolyadenylated RNA” comprising at least 20 consecutive nucle- otides having 95% sequence identity to the complement of the sequence of the target nucleic acid, such as described in WOOl/12824 or US6423885 (both documents herein incorporated by refer- ence).
  • RNA molecule as described in W003/076619 (herein incorporated by reference) comprising at least 20 consecutive nucleotides having 95% se- quence identity to the sequence of the target nucleic acid or the complement thereof, and further comprising a largely-double stranded region as described in W003/076619 (including largely dou- ble stranded regions comprising a nuclear localization signal from a viroid of the Potato spindle tuber viroid-type or comprising CUG trinucleotide repeats).
  • Silencing RNA may also be double stranded RNA comprising a sense and antisense strand as herein defined, wherein the sense and antisense strand are capable of base-pairing with each other to form a double stranded RNA region (preferably the said at least 20 consecutive nucleotides of the sense and antisense RNA are com- plementary to each other).
  • the sense and antisense region may also be present within one RNA molecule such that a hairpin RNA (hpRNA) can be formed when the sense and antisense region form a double stranded RNA region.
  • hpRNA hairpin RNA
  • the hpRNA may be classified as long hpRNA, having long, sense and antisense regions which can be largely complementary, but need not be entirely comple- mentary (typically larger than about 200 bp, ranging between 200-1000 bp). hpRNA can also be rather small ranging in size from about 30 to about 42 bp, but not much longer than 94 bp (see W004/073390, herein incorporated by reference). Silencing RNA may also be artificial micro- RNA molecules as described e.g. in W02005/052170, W02005/047505 or US 2005/0144667, or ta-siRNAs as described in W02006/074400 (all documents incorporated herein by reference).
  • RNA capable of modulating the expression of a gene can also be an RNA ribozyme.
  • operably linked refers to the functional spatial arrangement of two or more nu- cleic acid regions or nucleic acid sequences.
  • a promoter region may be positioned relative to a nucleic acid sequence such that transcription of a nucleic acid sequence is directed by the promoter region.
  • a promoter region is "operably linked” to the nucleic acid sequence. “Functionally linked” is an equivalent term.
  • A“transcription termination and polyadenylation region” as used herein is a sequence that controls the cleavage of the nascent RNA, whereafter a poly(A) tail is added at the resulting RNA 3’ end, functional in plant cells. Transcription termination and polyadenylation signals functional in plant cells include, but are not limited to, 3’nos, 3’35S, 3’his and 3’g7.
  • plant-expressible promoter means a DNA sequence that is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin such as the CaMV35S (Harpster et l. (198 a8) Mol Gen Genet.
  • the subterranean clover virus promoter No 4 or No 7 (WO9606932), or T-DNA gene promoters but also tissue-specific or organ-specific promoters including but not lim- ited to seed-specific promoters (e.g., WO89/03887), organ-primordia specific promoters (An et l. (1996) Plant Cell 8(1): 15-30), stem-specific promoters (Keller et l., (19 a88 ) EMBO J. 7(12): 3625- 3633), leaf specific promoters (Hudspeth et l. a (1989) Plant Mol Biol.
  • mesophyl- specific promoters such as the light-inducible Rubisco promoters
  • root-specific promoters such as the light-inducible Rubisco promoters
  • tuber-specific promoters such as the tuber-specific promoters
  • stamen- selective promoters WO 89/10396, WO 92/13956
  • dehiscence zone specific promoters WO 97/13865
  • Suitable promoters for the invention are constitutive plant-expressible promoters.
  • Constitutive plant-expressible promoters are well known in the art and include the CaMV35S promoter (Harp- ster et l a. (1988) Mol Gen Genet. 212(1): 182-90), Actin promoters, such as, for example, the pro- moter from the Rice Actin gene (McElroy et al., 1990, Plant Cell 2: 163), the promoter of the Cas- sava Vein Mosaic Virus (Verdaguer et al., 1996 Plant Mol. Biol. 31: 1129), the GOS promoter (de Pater et al., 1992, Plant J.
  • a further promoter suitable for the invention is the endogenous promoter driving expression of the gene encoding an Rca protein.
  • Any of the nucleic acid sequences described above may be provided in a recombinant vector.
  • a recombinant vector typically comprises, in a 5' to 3' orientation: a promoter to direct the tran- scription of a nucleic acid sequence and a nucleic acid sequence.
  • the recombinant vector may further comprise a 3' transcriptional terminator, a 3' polyadenylation signal, other untranslated nu- cleic acid sequences, transit and targeting nucleic acid sequences, selectable markers, enhancers, and operators, as desired.
  • 5' UTR refers to the untranslated region of DNA upstream, or 5' of the coding region of a gene and "3' UTR” refers to the untranslated region of DNA down- stream, or 3' of the coding region of a gene.
  • Means for preparing recombinant vectors are well known in the art. Methods for making recombinant vectors particularly suited to plant transfor- mation are described in US4971908, US4940835, US4769061 and US4757011. Typical vectors useful for expression of nucleic acids in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens .
  • Ti tumor-inducing
  • One or more addi- tional promoters may also be provided in the recombinant vector. These promoters may be operably linked, for example, without limitation, to any of the nucleic acid sequences described above. Al- ternatively, the promoters may be operably linked to other nucleic acid sequences, such as those encoding transit peptides, selectable marker proteins, or antisense sequences. These additional pro- moters may be selected on the basis of the cell type into which the vector will be inserted. Also, promoters which function in bacteria, yeast, and plants are all well taught in the art. The additional promoters may also be selected on the basis of their regulatory features. Examples of such features include enhancement of transcriptional activity, inducibility, tissue specificity, and developmental stage-specificity.
  • the recombinant vector may also contain one or more additional nucleic acid sequences.
  • additional nucleic acid sequences may generally be any sequences suitable for use in a recombinant vector. Such nucleic acid sequences include, without limitation, any of the nucleic acid sequences, and modified forms thereof, described above.
  • the additional structural nucleic acid sequences may also be operably linked to any of the above described promoters.
  • the one or more structural nucleic acid sequences may each be operably linked to separate promoters. Alternatively, the structural nucleic acid sequences may be operably linked to a single promoter (i.e. a single operon).
  • the invention provides a method for increasing the ratio of an arginine Rca a protein variant in plants comprising (a) providing to cells of a plant the recombinant gene of the invention; and reducing the expression of endogenous Rca a protein in said plant cells, wherein said ratio is increased compared to a control plant cell not comprising said recombinant gene; or (b) introducing into cells of a plant at least one arginine Rca a allele according to the invention, wherein said ratio is increased compared to a control plant cell not comprising said arginine Rca a allele allele.
  • the cereal plant is a wheat plant, a Brassica plant, a cotton plant, a maize plant, a soybean plant, a rice plant or an Arabidopsis plant.
  • said Rca a protein variants comprise an amino acid sequence as de- scribed above and is encoded by nucleic acids comprising the coding nucleic acid sequences de- scribed above.
  • Increasing the ratio of an arginine Rca a protein variant mean increasing the relative abundance of arginine Rca a protein variants over the overall abundance of Rca a proteins. This can be achieved by increasing the abundance of arginine Rca a protein variants, by decreasing the abundance of endogenous Rca a proteins and/or by both increasing the abundance of arginine Rca a protein variants and decreasing the abundance of endogenous Rca a proteins.
  • the increased ratio of arginine Rca a protein variants may be of at least about or about 15%, at least about or about 30%, at least about or about 45%, at least about or about 60%, at least about or about 75%, at least about or about 90%, or at least about or about 100%.
  • the increased ratio of arginine Rca a protein variants may be between about 15% and about 30%, between about 15% and about 45%, between about 15% and about 60%, between about 15% and about 75%, between about 15% and about 90%, between about 15% and about 100%, between about 30% and about 45%, between about 30% and about 60%, between about 30% and about 75%, between about 30% and about 90%, between about 30% and about 100%, between about 45% and about 60%, between about 45% and about 75%, between about 45% and about 90%, between about 45% and about 100%, between about 60% and about 75%, between about 60% and about 90%, between about 60% and about 100%, between about 75% and about 90%, between about 75% and about 100%, between about 90% and about 100%.
  • “Introducing” in connection with the present application relates to the placing of genetic in- formation in a plant cell or plant by artificial means. This can be effected by any method known in the art for introducing RNA or DNA into plant cells, protoplasts, calli, roots, tubers, seeds, stems, leaves, seedlings, embryos, pollen and microspores, other plant tissues, or whole plants. "Introduc- ing” also comprises stably integrating into the plant's genome. Introducing the recombinant gene can be performed by transformation or by crossing with a plant obtained by transformation or its descendant (also referred to as“introgression”). Introducing an allele also may be performed by mutagenesis of by gene editing.
  • the term“providing” may refer to introduction of an exogenous DNA molecule to a plant cell by transformation, optionally followed by regeneration of a plant from the transformed plant cell.
  • the term may also refer to introduction of the recombinant DNA molecule by crossing of a trans- genic plant comprising the recombinant DNA molecule with another plant and selecting progeny plants which have inherited the recombinant DNA molecule or transgene.
  • Yet another alternative meaning of providing refers to introduction of the recombinant DNA molecule by techniques such as protoplast fusion, optionally followed by regeneration of a plant from the fused protoplasts.
  • the recombinant gene may be provided to a plant cell by methods well-known in the art.
  • transformation herein refers to the introduction (or transfer) of nucleic acid into a recipient host such as a plant or any plant parts or tissues including plant cells, protoplasts, calli, roots, tubers, seeds, stems, leaves, fibers, seedlings, embryos and pollen. Plants containing the transformed nucleic acid sequence are referred to as“transgenic plants”. Transformed, transgenic and recombinant refer to a host organism such as a plant into which a heterologous nucleic acid molecule (e.g. an expression cassette or a recombinant vector) has been introduced. The nucleic acid can be stably integrated into the genome of the plant.
  • a heterologous nucleic acid molecule e.g. an expression cassette or a recombinant vector
  • transgenic plant refers to a plant having a nucleic acid stably integrated into a genome of the plant, for example, the nuclear or plastid genomes.
  • plants containing transformed nucleic acid sequence are referred to as “transgenic plants” and in- cludes plants directly obtained from transformation and their descendants (Tx generations).
  • Trans- genic and recombinant refer to a host organism such as a plant into which a heterologous nucleic acid molecule (e.g. the promoter, the recombinant gene or the vector as described herein) has been introduced.
  • the nucleic acid can be stably integrated into the genome of the plant.
  • Transformation of plants is now a routine technique.
  • any of several transformation methods may be used to introduce the nucleic acid/gene of interest into a suitable ancestor cell. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens et al. (1982) Nature 296: 72-74 ; Negru- tiu et al. (1987) Plant. Mol. Biol.
  • Control plant refers to a plant genetically resembling the tested plant but not carrying the recombinant gene, such as wild type plants or null segregant plants, or not carrying the mutant allele, such as wild type plants or wild type segregant plants.
  • the transformed plant cells and plants obtained by the methods described herein may be fur- ther used in breeding procedures well known in the art, such as crossing, selfing, and backcrossing. Breeding programs may involve crossing to generate an FI (first filial) generation, followed by several generations of selfing (generating F2, F3, etc). The breeding program may also involve backcrossing (BC) steps, whereby the offspring is backcrossed to one of the parental lines, termed the recurrent parent.
  • transformed plant cells and plants obtained by the methods disclosed herein may also be further used in subsequent transformation procedures, e. g. to introduce a further recombinant gene.
  • reducing the expression of endogenous Rca proteins comprises in- troducing into cells of the plant at least one knock-out mutant Rca allele according to the invention, or providing said cells of a plant with a second recombinant gene capable of suppressing specifi- cally the expression of the endogenous Rca a gene, as described above.
  • Suitable for the invention are methods for increasing the ratio of an arginine Rca a protein variants in plants comprising introducing into cells of a wheat plant at least two, at least three, at least four, at least five or even all six knock-out mutant Rca a allele s according to the invention.
  • Such at least two knock-out mutant Rca a allele s in wheat may be two knock-out mutant Rca a alleles from the subgenome B, two knock-out mutant Rca a allele s from the subgenome D, two knock-out mutant Rca a allele s from the subgenome A, one knock-out mutant Rca a allele from the subgenome B and one knock-out mutant Rca a allele from the subgenome D, one knock-out mutant Rca a allele from the subgenome B and one knock-out mutant Rca a allele from the subge- nome A or one knock-out mutant Rca a allele from the subgenome D and one knock-out mutant Rca a allele from the subgenome A.
  • Such at least three knock-out mutant Rca a allele s in wheat may be two knock-out mutant Rca a alleles from the subgenome B and one knock-out mutant Rca a allele from the subgenome A, two knock-out mutant Rca a allele s from the subgenome B and one knock-out mutant Rca a allele from the subgenome D, two knock-out mutant Rca a allele s from the subgenome D and one knock-out mutant Rca a allele from the subgenome B, two knock- out mutant Rca a allele s from the subgenome D and one knock-out mutant Rca a allele from the subgenome A, two knock-out mutant Rca a alleles from the subgenome A and one knock-out mu- tant Rca a allele from the subgenome B, two knock-out mutant Rca a alleles from the subgenome A and one knock-out mutant Rca a allele from the subgenome D or one knock-out mutant Rca
  • Such at least four knock-out mutant Rca a alleles in wheat may be two knock-out mutant Rca a alleles from the subgenome B and two knock-out mutant Rca a allele s from the subgenome A, two knock-out mutant Rca a alleles from the subgenome B and two knock-out mutant Rca a allele from the subgenome D, or two knock-out mutant Rca a allele s from the subgenome D and two knock-out mutant Rca a allele from the subgenome A.
  • Such at least four knock-out mutant Rca a alleles may also be two knock-out mutant Rca a allele s from the subgenome B, one knock-out mutant Rca a allele s from the subgenome A and one knock-out mutant Rca a allele s from the subgenome D, or two knock-out mutant Rca alle ales from the subgenome D, one knock-out mutant Rca a alleles from the subgenome A and one knock-out mutant Rca a alleles from the subgenome B, or two knock-out mutant Rca a allele s from the subgenome A, one knock-out mutant Rca a alleles from the subgenome B and one knock-out mutant Rca allel aes from the subgenome D.
  • Such at least five knock-out mutant Rca a allele s may be two knock-out mutant Rca a allele s from the subgenome B, two knock-out mutant Rca a alleles from the subgenome A and one knock-out mu- tant Rca a allele from the subgenome D, or two knock-out mutant Rca allele as from the subgenome B, two knock-out mutant Rca a allele s from the subgenome D and one knock-out mutant Rca a allele from the subgenome A, or two knock-out mutant Rca a allele s from the subgenome D, two knock-out mutant Rca a alleles from the subgenome A and one knock-out mutant Rca allele a from the subgenome B.
  • Suitable for the invention are methods for increasing the ratio of an arginine Rca a protein variants in plants comprising introducing into cells of a Brassica plant at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or even all twelve knock-out mutant Rca a alleles according to the invention.
  • Such at least two knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 alleles from the subgenome A, two knock-out mutant Rca 2 alleles from the subgenome A, two knock-out mutant Rca 3 alleles from the subgenome A, two knock-out mutant Rca 1 alleles from the subgenome C, two knock-out mutant Rca 2 alleles from the subgenome C, two knock-out mu- tant Rca 3 alleles from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca
  • Such at least three knock-out mutant in Brassica may be two knock-out mutant Rca 1 alleles from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A, two knock- out mutant Rca 1 alleles from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 alleles from the subgenome A and one knock-out mu- tant Rca 1 allele from the subgenome C, two knock-out mutant Rca 1 alleles from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 1 alleles from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 2 alleles from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 alleles from the subgenome A and one knock-out mutant Rca 1 allele from
  • Such at least three knock-out mutant in Brassica may also be one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A , one knock-out mutant Rca 1 allele from the subgenome A , one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and
  • Such at least four knock-out mutant Rca alleles in Brassica may be one knock-out mutant Rca
  • Such at least four knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgen
  • Such at least four knock-out mutant Rca alleles in Brassica may furthermore be two knock- out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subge- nome C, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 1
  • Such at least five knock-out mutant Rca alleles in Brassica may be one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome
  • Such at least five knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgen
  • Such at least five knock-out mutant Rca alleles in Brassica may furthermore be two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome A and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the sub
  • Such at least six knock-out mutant Rca alleles in Brassica may be one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C,one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C.
  • Such at least six knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgen
  • Such at least six knock-out mutant Rca alleles in Brassica may furthermore be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele to the sub
  • Such at least six knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgen
  • Such at least seven knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome
  • Such at least seven knock-out mutant Rca alleles in Brassica may also be two knock-out mu- tant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca
  • Such at least seven knock-out mutant Rca alleles in Brassica may even be two knock-out mu- tant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the
  • Such at least eight knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subge- nome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the sub
  • Such at least eight knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the
  • Such at least eight knock-out mutant Rca alleles in Brassica may furthermore be two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome A, two knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub
  • Such at least nine knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome
  • Such at least nine knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgen
  • Such at least ten knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgen
  • Such at least ten knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub
  • Such at least eleven knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome C and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subge- nome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub
  • Such at least twelve knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C.
  • Suitable for the invention are methods for increasing the ratio of an arginine Rca a protein variants in plants comprising introducing into cells of a cotton plant at least two, at least three, at least four, at least five, at least six, at least seven or even all eight knock-out mutant Rca a alleles according to the invention.
  • Such at least two knock-out mutant Gossypium Rca alleles may be one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the
  • Such at least three knock-out mutant Gossypium Rca alleles may be one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the
  • Such at least four knock-out mutant Gossypium Rca alleles may be one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the
  • Such at least five knock-out mutant Gossypium Rca alleles may be two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome D, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome D, one knock-out mutant Rca 1 allele from the
  • Such at least six knock-out mutant Gossypium Rca alleles may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome D, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the
  • Such at least seven knock-out mutant Gossypium Rca alleles may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome D and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D and two knock-out mutant Rca 2 allele from the subgenome D; one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the
  • Such at least eight knock-out mutant Gossypium Rca alleles may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D and two knock-out mutant Rca 2 allele from the subgenome D.
  • Suitable for the invention are methods for increasing the ratio of an arginine Rca a protein variants in plants comprising introducing into cells of a soybean plant at least two, at least three, at least four, at least five, or even all six knock-out mutant Rca a alleles according to the invention.
  • Such at least two knock-out mutant soybean Rca alleles may be two knock-out mutant Rca 1 allele; two knock-out mutant Rca 2 allele, two knock-out mutant Rca 3 allele; one knock-out mutant Rca 1 allele and one knock-out mutant Rca 2 allele; one knock-out mutant Rca 1 allele andf one knock-out mutant Rca 3 allele; one knock-out mutant Rca 2 allele and one knock-out mutant Rca 3 allele.
  • Such at least three knock-out mutant soybean Rca alleles may be one knock-out mutant Rca 1 allele, one knock-out mutant Rca 2 allele and one knock-out mutant Rca 3 allele; two knock- out mutant Rca 1 allele and one knock-out mutant Rca 2 allele; two knock-out mutant Rca 1 allele and one knock-out mutant Rca 3 allele; two knock-out mutant Rca 2 allele and one knock-out mutant Rca 1 allele; two knock-out mutant Rca 2 allele and one knock-out mutant Rca 3 allele; two knock-out mutant Rca 3 allele and one knock-out mutant Rca 1 allele; two knock-out mutant Rca 3 allele and one knock-out mutant Rca 2 allele.
  • Such at least four knock-out mutant soybean Rca alleles may be two knock-out mutant Rca 1 allele, one knock-out mutant Rca 2 allele and one knock-out mutant Rca 3 allele; two knock-out mutant Rca 2 allele, one knock-out mutant Rca 1 allele and one knock-out mutant Rca 3 allele; two knock-out mutant Rca 3 allele, one knock-out mutant Rca 1 allele and one knock-out mutant Rca 2 allele; two knock-out mutant Rca 1 allele and two knock-out mutant Rca 2 allele; two knock-out mutant Rca 1 allele and two knock-out mutant Rca 3 allele; two knock-out mutant Rca 2 allele and two knock-out mutant Rca 3 allele.
  • Such at least five knock-out mutant soybean Rca alleles may be two knock-out mutant Rca 1 allele, two knock-out mutant Rca 2 allele and one knock-out mutant Rca 3 allele; two knock-out mutant Rca 1 allele, two knock-out mutant Rca 3 allele and one knock- out mutant Rca 2 allele; two knock-out mutant Rca 2 allele, two knock-out mutant Rca 3 allele and one knock-out mutant Rca 1 allele.
  • Such at least six knock-out mutant soybean Rca alleles may be two knock-out mutant Rca 1 allele, two knock-out mutant Rca 2 allele and two knock-out mutant Rca 3 allele.
  • reducing the expression of endogenous Rca a protein in plant cells refers to a reduction in the amount of a functional Rca a protein produced by the cell comprising the at least one knock- out mutant Rca allele according to the invention or the second recombinant gene capable of sup- pressing specifically the expression of the endogenous Rca gene as described above, by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% as compared to the amount of the functional Rca a protein produced by the cells not comprising the at least one knock-out mutant Rca allele according to the invention or the second recombinant gene capable of suppressing spe- cifically the expression of the endogenous Rca a gene as described above.
  • Also suitable for the invention are methods for increasing the ratio of an arginine Rca a protein variant in wheat comprising introducing into cells of the cereal plant at least two, at least three, at least four, at least five or even all six arginine Rca a alleles according to the invention, methods for increasing the ratio of an arginine Rca a protein variant in Brassica comprising introducing into cells of the Brassica plant at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or even all twelve arginine Rca a alleles according to the invention, methods for increasing the ratio of an arginine Rca a protein variant in cotton comprising introducing into cells of the cotton plant at least two, at least three, at least four, at least five, at least six, at least seven or even all eight arginine Rca a alleles according to the invention, methods for increasing the ratio of an arginine Rca a protein variant in soybean
  • a method for increasing the photosynthetic activity of a plant which comprises increasing the ratio of an arginine Rca protein variant and regenerating said plant, wherein the photosynthetic activity is increased compared to a plant not comprising said increased ratio of an arginine Rca a protein variant.
  • the ratio of an arginine Rca a protein variant is increased according to the method for increasing the ratio of an arginine Rca a protein variant in plants described herein.
  • said arginine Rca a protein var- iant is the Rca a protein variant according to the invention.
  • the arginine Rca a protein variant may be a variant of an Rca a protein originating from a dicotyle- donous plant.
  • the arginine Rca a protein variant may be a variant of an Rca a protein originating from a monocotyledonous plant.
  • the arginine Rca a protein variant may be a variant of an Rca a protein originating from the same species or a crossable species as the plant for which the photosynthetic activity is to be increased.
  • the photosynthetic activity of a plant can be measured by methods well known in the art (see for example Kalaji et al 2012 Photosynth Res 114:69-96 or Long and Bemacchi, JEB 2003).
  • a method for increasing yield of a plant comprising increasing the ratio of an arginine Rca a protein variant and regenerating said plant, wherein the yield is increased compared to a plant not comprising said increased ratio of an arginine Rca a protein variant.
  • the ratio of an arginine Rca a protein variant is increased according to the method for increasing the ratio of an arginine Rca a protein variant in plants described herein.
  • said arginine Rca a protein variant is the arginine Rca a protein variant according to the invention.
  • the yield increased may be seed yield or thousand seed weight.
  • the arginine Rca a protein variant may be a variant of an Rca a protein originating from a dicotyledonous plant.
  • the arginine Rca a protein variant may be a variant of an Rca a protein originating from a mono- cotyledonous plant.
  • the arginine Rca a protein variant may be a variant of an Rca a protein origi- nating from the same species or a crossable species as the plant for which the yield is to be in- creased.
  • a method for producing a plant such as a wheat plant, a cotton plant, a brassica plant, a rice plant, a soybean plant, a maize plant, with increased photosynthetic activity and/ or an increased yield is furthermore provided, comprising increasing the ratio of an arginine Rca a protein variant as disclosed herein and regenerating said plant.
  • arginine Rca a protein variant according to the invention the nucleic acid encoding an arginine Rca a protein variant according to the invention, the recombinant gene according to the invention, the recombinant gene capable of suppressing specifically the ex- pression of the endogenous Rca genes described herein or the arginine allele of a Rca a gene pro- vided herewith to increase the ratio of an arginine Rca a protein variant in plants, to increase photosynthetic activity of a plant, to increase yield of a plant or to produce a plant with increased rubisco activity and/ or yield.
  • Such plant may be a wheat plant, a Brassica plant, a cotton plant, a rice plant, a maize plant or a soybean plant.
  • Yield as used herein can comprise yield of the plant or plant part which is harvested, such as biomass, or seed, including seed protein content and seed oil content, seed weight (measured as thousand seed weigth), seed number. Increased yield can be increased yield per plant and/or in- creased yield per surface unit of cultivated land, such as yield per hectare.
  • the yield increase achieved with the method described herein compared to plants wherein the ratio of an arginine Rca a protein variant is not increased may be of at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9% or at least about 10%.
  • the yield increase achieved with the method described herein compared to plants wherein the ratio of an arginine Rca a protein variant is not increased may be of at least about 5%, at least about 6%, at least about 7% or at least about 8%, at least about 9% or at least about 10%.
  • Another aspect of the invention provides a method of producing food, feed, such as meal, grain, starch, flour or protein, or an industrial product, such as biofuel, fiber, industrial chemicals, a pharmaceutical or a nutraceutical, said method comprising obtaining the plant according to the invention or a part thereof, and preparing the food, feed or industrial product from the plant or part thereof.
  • examples of food products include flour, starch, leavened or unleavened breads, pasta, noodles, animal fodder, breakfast cereals, snack foods, cakes, malt, pastries, instantan and foods containing flour-based sauces.
  • the flour is produced by grinding finely grains in a mill (see for example www.madehow.com/Volume-3/Flour.html) and the biofuel is produced from wheat straw or mix- tures of wheat straw and wheat meal (see for example Erdei et al., Biotechnology for Biofuels, 2010, 3: 16).
  • a method of increasing the maximal Rubisco activase activity and/or reducing the ADP inhibition of the Rubisco activase activity of a Rca a protein comprising introducing an amino acid substitution to the amino acid sequence of said Rca a protein, wherein the amino acid substitution is substituting or replacing an amino acid with an arginine at a position corresponding to position 382 of SEQ ID NO: 7, wherein the maximal Rubisco activase activity of a Rca a protein is increased compared to the maximal Rubisco activase activity of the Rubisco activase activity of a Rca a protein not comprising the amino acid substitution and/or wherein the ADP inhibition of the Rubisco activase activity of a Rca a protein is reduced compared to the ADP inhibition of the Rubisco activase activity of a Rca a protein not comprising the amino acid substitution.
  • Suitable for the invention are increases in maximal Rubisco activase activity of the Rca a protein comprising said amino acid substitution by at least about or about 3%, at least about or about 4%, at least about or about 5%, at least about or about 6%, at least about or about 7%, at least about or about 8%, at least about or about 9%, at least about or about 10%.
  • Suitable for the invention are reductions in ADP inhibition of the Rubisco activase activity of the Rca a protein comprising said amino acid substitution by at least about or about 3%, at least about or about 4%, at least about or about 5%, at least about or about 6%, at least about or about 7%, at least about or about 8%, at least about or about 9%, at least about or about 10%.
  • a method for producing an arginine Rca a protein variant is herein provided, com- prising culturing the host cell comprising the recombinant gene comprising the nucleic acid encod- ing an arginine Rca a protein variant as described above and isolating the protein produced.
  • Said host cell expresses or over-expresses the arginine Rca protein variant of the invention.
  • said protein of the invention is produced in and isolated from the host cell.
  • the host cell produces the protein of the invention and secretes it to the surrounding media, e. g. due to a suitable signal peptide attached to the protein
  • isolation denotes separation of the media comprising the protein from the host cell.
  • Said media may then be the subject of further purification steps (see below).
  • Suitable conditions for culturing a prokaryotic or eukaryotic host are well known to the person skilled in the art.
  • suitable conditions for culturing bacteria are growing them under aeration in Luria Bertani (LB) medium.
  • the medium can be buffered or supplemented with suitable additives known to enhance or facilitate both.
  • E. coli can be cultured from 4 to about 37°C, the exact temperature or sequence of temperatures depends on the molecule to be over-expressed. In general, the skilled person is also aware that these conditions may have to be adapted to the needs of the host and the requirements of the polypeptide expressed.
  • Suitable expression protocols and strategies are known to the skilled person.
  • Suitable expression protocols for eukaryotic cells are well known to the skilled person and can be retrieved e.g. from Sambrook, 2001.
  • Suitable media for insect cell culture are e.g. TNM + 10% FCS or SF900 medium. Insect cells are usually grown at 27°C as adhesion or suspension culture.
  • Methods of isolation of the polypeptide produced are well-known in the art and comprise with- out limitation method steps such as ammonium sulphate precipitation, ion exchange chromatog- raphy, gel filtration chromatography (size exclusion chromatography), affinity chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC, disc gel electrophoresis or immunoprecipitation, see, for example, in Sambrook, 2001.
  • a host cell such as an E. coli cell, an Agrobacterium cell, a yeast cell, or a plant cell, comprising (a) the recombinant gene comprising a nucleic acid encoding an arginine Rca a protein variant according to the invention or the vector comprising this recombinant gene, (b) the recombinant gene capable of suppressing specifically the expression of the endoge- nous Rca a genes as described herein or the vector comprising this recombinant gene, or (c) the arginine Rca a protein variant according to the invention.
  • a host cell such as an E. coli cell, an Agrobacterium cell, a yeast cell, or a plant cell, comprising (a) the recombinant gene comprising a nucleic acid encoding an arginine Rca a protein variant according to the invention or the vector comprising this recombinant gene, (b) the recombinant gene capable of suppressing specifically the expression of the endoge
  • a plant cell comprising (a) at least one knock-out Rca a allele as described herein and/or (b) at least one arginine Rca a allele according to the invention.
  • the plant cell comprising the recombinant gene comprising a nucleic acid en- coding an arginine Rca a protein variant according to the invention or the vector comprising this recombinant gene may further comprise the recombinant gene capable of suppressing specifically the expression of the endogenous Rca a genes as described herein or the vector comprising that recombinant gene or at least one knock-out Rca a allele as described herein.
  • the plant cell may be a cereal plant cell, a wheat plant cell, a com plant cell, a cotton plant cell, a Brassica plant cell, a rice plant cell or a soybean plant cell.
  • a plant that comprises the arginine Rca a protein variant according to the invention.
  • Said plant may be a cereal plant, a wheat plant, a com plant, a cotton plant, a Brassica plant, a rice plant or a soybean plant.
  • nucleic acid sequences may also be introduced into the host cell along with the described recombinant genes described herein, e. g. also in connection with the vector of the invention. These other sequences may include 3' transcriptional terminators, 3' polyadenylation signals, other un- translated nucleic acid sequences, transit or targeting sequences, selectable markers, enhancers, and operators.
  • Preferred nucleic acid sequences of the present invention including recombinant vectors, structural nucleic acid sequences, promoters, and other regulatory elements, are described above.
  • a plant comprising any of the recombinant genes and alleles according to the invention.
  • a further embodiment provides plant parts and seeds obtainable from the plant according to the invention. These plant parts and seeds comprise the recombinant genes or alleles described above.
  • the plants, plant parts or seeds according to the invention are wheat plants, plant parts or seeds, Brassica plants, plant parts or seeds, rice plants, plant parts or seeds, cotton plants, plant parts or seeds, soybean plants, plant parts or seeds or com plants, plant parts or seeds.
  • Plants encompasses“monocotyledonous plants” and“dicotyledonous plants”.
  • “Monocotyledonous plants”, also known as“monocot plants” or“monocots” are well known in the art and are plants of which the seed typically has one cotyledon.
  • Examples of monocotyle- dons plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lo- lium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, triticale, spelt, einkom, emmer, durum wheat, kamut, sorghum, and maize (com).
  • the plants according to the invention may be cereal plants.
  • the cereal plants according to the invention may be wheat plants, rice plants or com plants.
  • “Wheat” or“wheat plant” as used herein can be any variety useful for growing wheat. Exam- ples of wheat are, but are not limited to, Triticum aestivum, Triticum aethiopicum, Triticum Com- pactum, Triticum dicoccoides, Triticum dicoccon, Triticum dumm, Triticum monococcum, Triti- cum spelta, Triticum turgidum.“Wheat” furthermore encompasses spring and winter wheat varie- ties, with the winter wheat varieties being defined by a vernalization requirement to flower while the spring wheat varieties do not require such vernalization to flower.
  • “Com”,“maize”“maize plant” or“com plant” as used herein can be any variety useful for growing com.
  • the six major types of maize (Zea mays) are dent com (Zea Mays var. indentata), flint com (Zea Mays var. indurata), pod com (Zea Mays var. tunicata), popcorn (Zea Mays var. everta), flour com (Zea Mays var. amylacea), and sweet com (Zea Mays convar. Saccharata var. mgosa).
  • Rice contains two major subspecies: short-grained japonica or sinica varieties and the long- grained indica variety. A third subspecies which is broadgrained and thrives under tropical condi- tions was initially referred to as javanica, but is now known as tropical japonica. Whenever "rice” is used herein, it will be understood that all three subspecies are included.
  • “Dicotyledonous plants”, also known as“dicot plants” or“dicots” are well known in the art and are plants of which the seed typically has two cotyledons. Examples of families of dicotyle- donous plants are Brassicaceae, Solanaceae, Fabaceae, Malvaceae.
  • Brassicaceae or“Brassicaceae plant” as used herein refers to plants belonging to the family of Brassicaceae plants, also called Cruciferae or mustard family.
  • Brassicaceae are, but are not limited to, Brassica species, such as Brassica napus, Brassica oleracea, Brassica rapa, Brassica carinata, Brassica nigra, and Brassica juncea; Raphanus species, such as Raphanus cau- datus, Raphanus raphanistrum, and Raphanus sativus; Matthiola species; Cheiranthus species; Camelina species, such as Camelina sativa; Cramhe species, such as Cramhe b ayssinica and Crambe hispanica; Eruca species, such as Eruca vesicaria; Sinapis species such as Sinapis lba a; Diplotaxis species; Lepidium species; Nasturtium species; Orychophragmus species; Armoracia
  • Malvaceae refers to plants belonging to the family of Malvaceae plants, also called mallows family.
  • Malvaceae are, but are not limited to, Gossypium species, such as Gossypium hirsutum, Gossypium barbadense, Gossypium rbor aeum and Gossypium herbaceum or progeny from crosses of such species with other species or crosses between such species.
  • Cotton or“cotton plant” as used herein can be any variety useful for growing cotton.
  • the most commonly used cotton varieties are Gossypium barbadense, G. hirsutum, G. rbo areum and G. herbaceum. Further varieties include G. fric aanum and G. raimondii. Also included are progeny from crosses of any of the above species with other species or crosses between such species.
  • Fabaceae refers to the plant commonly known as the legume, pea, or bean family plants. Examples of Fabaceae are, but are not limited to, Glycine max (soybean), Phaseolus (beans), Pisum sativum (pea), Cicer arietinum (chickpeas), Medicago sativa (alfalfa), Arachis hy- pogaea (peanut), Lathyrus odoratus (sweet pea), Ceratonia siliqua (carob), and Glycyrrhiza glabra (liquorice).
  • Plant parts are parts of the plant, which can be cells, tissues or organs, such as seeds, severed parts such as roots, leaves, flowers, pollen, etc.
  • the plants according to the invention may additionally contain an endogenous or a transgene, which confers herbicide resistance, such as the bar or pat gene, which confer resistance to glufosinate ammonium (Liberty®, Basta® or Ignite®) [EP 0 242 236 and EP 0 242 246 incorpo- rated by reference]; or any modified EPSPS gene, such as the 2mEPSPS gene from maize [EP0 508 909 and EP 0 507 698 incorporated by reference], or glyphosate acetyltransferase, or glypho- sate oxidoreductase, which confer resistance to glyphosate (RoundupRcady®), or bromoxynitril nitrilase to confer bromoxynitril tolerance, or any modified AHAS gene, which confers tolerance to sulfonylureas, imidazolinones, sulfonylaminocarbonyl
  • the plants or seeds of the plants according to the invention may be further treated with a chemical compound, such as a chemical compound selected from the following lists: Herbicides: Clethodim, Clopyralid, Diclofop, Ethametsulfiiron, Fluazifop, Glufosinate, Glypho- sate, Metazachlor, Quinmerac, Quizalofop, Tepraloxydim, Trifluralin.
  • a chemical compound such as a chemical compound selected from the following lists: Herbicides: Clethodim, Clopyralid, Diclofop, Ethametsulfiiron, Fluazifop, Glufosinate, Glypho- sate, Metazachlor, Quinmerac, Quizalofop, Tepraloxydim, Trifluralin.
  • Fungicides / PGRs Azoxystrobin, N-[9-(dichloromethylene)-l,2,3,4-tetrahydro-l,4-meth- anonaphthalen-5 -yl] -3 -(difluoromethyl)- 1 -methyl- lH-pyrazole-4-carboxamide (Ben- zovindiflupyr, Benzodiflupyr), Bixafen, Boscalid, Carbendazim, Carboxin, Chlormequat-chloride, Coniothryrium minitans, Cyproconazole, Cyprodinil, Difenoconazole, Dimethomorph, Dimoxystrobin, Epoxiconazole, Famoxadone, Fluazinam, Fludioxonil, Fluopicolide, Fluopyram, Fluoxastrobin, Fluquinconazole, Flusilazole, Fluthianil, Flutriafol, Fluxapyrox
  • Insecticides Acetamiprid, Aldicarb, Azadirachtin, Carbofuran, Chlorantraniliprole (Rynaxypyr), Clothianidin, Cyantraniliprole (Cyazypyr), (beta-)Cyfluthrin, gamma-Cyhalothrin, lambda- Cyhalothrin, Cypermethrin, Deltamethrin, Dimethoate, Dinetofuran, Ethiprole, Flonicamid, Flubendiamide, Fluensulfone, Fluopyram, Flupyradifurone, tau-Fluvalinate, Imicyafos, Imidaclo- prid, Metaflumizone, Methiocarb, Pymetrozine, Pyrifluquinazon, Spinetoram, Spinosad, Spiro- tetramate, Sulfoxaflor, Thiacloprid, Thiamethoxam,
  • the plant cells of the invention as well as plant cells generated according to the methods of the invention may be non-propagating cells.
  • the obtained plants according to the invention can be used in a conventional breeding scheme to produce more plants with the same characteristics or to introduce the same characteristic in other varieties of the same or related plant species, or in hybrid plants.
  • the obtained plants can further be used for creating propagating material.
  • Plants according to the invention can further be used to produce gametes, seeds, embryos, either zygotic or somatic, progeny or hybrids of plants obtained by methods of the invention. Seeds obtained from the plants according to the invention are also encompassed by the invention.
  • Creating propagating material relates to any means know in the art to pro- prise further plants, plant parts or seeds and includes inter alia vegetative reproduction methods (e.g. air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, twin-scaling), sexual repro- duction (crossing with another plant) and asexual reproduction (e.g. apomixis, somatic hybridiza- tion).
  • vegetative reproduction methods e.g. air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, twin-scaling
  • sexual repro- duction crossing with another plant
  • asexual reproduction e.g. apomixis, somatic hybridiza- tion
  • plants according to the invention which however have been obtained exclusively by essentially biological processes, wherein a process for the production of plants is considered essentially biological if it consists entirely of natural phenomena such as crossing or selection, may be excluded from patentability. Plants according to the invention thus also encom- pass those plants not exclusively obtained by essentially biological processes.
  • SEQ ID NO: 1 amino acid sequence of the TaRca 2alpha from the subgenome
  • SEQ ID NO: 2 nucleotide sequence of the TaRca 2alpha from the subgenome
  • SEQ ID NO: 3 amino acid sequence of the TaRca 2alpha from the subgenome A minus the signal pep- tide
  • SEQ ID NO: 4 nucleotide sequence of the TaRca 2alpha from the subgenome A minus the signal peptide
  • SEQ ID NO: 5 amino acid sequence of the TaRca 2alpha from the subgenome B
  • SEQ ID NO: 6 nucleotide sequence of the TaRca 2alpha from the subgenome B
  • SEQ ID NO: 7 amino acid sequence of the TaRca 2alpha from the subgenome B minus the signal peptide
  • SEQ ID NO: 8 nucleotide sequence of the TaRca 2alpha from the subgenome B minus the signal peptide
  • SEQ ID NO: 9 amino acid sequence of the TaRca 2alpha from the subgenome D
  • SEQ ID NO: 10 nucleotide sequence of the TaRca 2alpha from the subgenome D
  • SEQ ID NO: 11 amino acid sequence of the TaRca 2alpha from the subgenome D minus the signal pep- tide
  • SEQ ID NO: 12 nucleotide sequence of the TaRca 2alpha from the subgenome D minus the signal pep- tide
  • SEQ ID NO: 13 amino acid sequence of the TaRca 1 beta from the subgenome A
  • SEQ ID NO: 14 nucleotide sequence of the TaRca 1 beta from the subgenome A
  • SEQ ID NO: 15 amino acid sequence of the TaRca 1 beta from the subgenome A minus the signal pep- tide
  • SEQ ID NO: 16 nucleotide sequence of the TaRca 1 beta from the subgenome A minus the signal pep- tide
  • SEQ ID NO: 17 amino acid sequence of the TaRca 1 beta from the subgenome B
  • SEQ ID NO: 18 nucleotide sequence of the TaRca 1 beta from the subgenome B
  • SEQ ID NO: 19 amino acid sequence of the TaRca 1 beta from the subgenome B minus the signal pep- tide
  • SEQ ID NO: 20 nucleotide sequence of the TaRca 1 beta from the subgenome B minus the signal peptide
  • SEQ ID NO: 21 amino acid sequence of the TaRca 1 beta from the subgenome D
  • SEQ ID NO: 22 nucleotide sequence of the TaRca 1 beta from the subgenome D
  • SEQ ID NO: 23 amino acid sequence of the TaRca 1 beta from the subgenome D minus the signal pep- tide
  • SEQ ID NO: 24 nucleotide sequence of the TaRca 1 beta from the subgenome D minus the signal pep- tide
  • SEQ ID NO: 25 amino acid sequence of the OsRca alpha
  • SEQ ID NO: 26 nucleotide sequence of the OsRca alpha
  • SEQ ID NO: 27 amino acid sequence of the OsRca alpha minus the signal peptide
  • SEQ ID NO: 28 nucleotide sequence of the OsRca alpha minus the signal peptide
  • SEQ ID NO: 29 amino acid sequence of the BnRca lalpha from the subgenome A
  • SEQ ID NO: 30 nucleotide sequence of the BnRca lalpha from the subgenome A
  • SEQ ID NO: 31 amino acid sequence of the BnRca lalpha from the subgenome A minus the signal pep- tide
  • SEQ ID NO: 32 nucleotide sequence of the BnRca lalpha from the subgenome A minus the signal pep- tide
  • SEQ ID NO: 33 amino acid sequence of the BnRca 2alpha from the subgenome A
  • SEQ ID NO: 34 nucleotide sequence of the BnRca 2alpha from the subgenome A
  • SEQ ID NO: 35 amino acid sequence of the BnRca 2alpha from the subgenome A minus the signal pep- tide
  • SEQ ID NO: 36 nucleotide sequence of the BnRca 2alpha from the subgenome A minus the signal pep- tide
  • SEQ ID NO: 37 amino acid sequence of the BnRca 3 alpha from the subgenome A
  • SEQ ID NO: 38 nucleotide sequence of the BnRca 3 alpha from the subgenome A
  • SEQ ID NO: 39 amino acid sequence of the BnRca 3 alpha from the subgenome A minus the signal pep- tide
  • SEQ ID NO: 40 nucleotide sequence of the BnRca 3 alpha from the subgenome A minus the signal pep- tide
  • SEQ ID NO: 41 amino acid sequence of the BnRca lalpha from the subgenome C
  • SEQ ID NO: 42 nucleotide sequence of the BnRca lalpha from the subgenome C
  • SEQ ID NO: 43 amino acid sequence of the BnRca lalpha from the subgenome C minus the signal pep- tide
  • SEQ ID NO: 44 nucleotide sequence of the BnRca lalpha from the subgenome C minus the signal pep- tide
  • SEQ ID NO: 45 amino acid sequence of the BnRca 2alpha from the subgenome C
  • SEQ ID NO: 46 nucleotide sequence of the BnRca 2alpha from the subgenome C
  • SEQ ID NO: 47 amino acid sequence of the BnRca 2alpha from the subgenome C minus the signal pep- tide
  • SEQ ID NO: 48 nucleotide sequence of the BnRca 2alpha from the subgenome C minus the signal pep- tide
  • SEQ ID NO: 49 amino acid sequence of the BnRca 3 alpha from the subgenome C
  • SEQ ID NO: 50 nucleotide sequence of the BnRca 3 alpha from the subgenome C
  • SEQ ID NO: 51 amino acid sequence of the BnRca 3from the subgenome C minus the signal peptide
  • SEQ ID NO: 52 nucleotide sequence of the BnRca 3from the subgenome C minus the signal peptide
  • SEQ ID NO: 53 amino acid sequence of the GhRca 1 alpha from the subgenome A
  • SEQ ID NO: 54 nucleotide sequence of the GhRca 1 alpha from the subgenome A
  • SEQ ID NO: 55 amino acid sequence of the GhRca 1 alpha from the subgenome A minus the signal pep- tide
  • SEQ ID NO: 56 nucleotide sequence of the GhRca 1 alpha from the subgenome A minus the signal pep- tide
  • SEQ ID NO: 57 amino acid sequence of the GhRca 2 alpha from the subgenome A
  • SEQ ID NO: 58 nucleotide sequence of the GhRca 2 alpha from the subgenome A
  • SEQ ID NO: 59 amino acid sequence of the GhRca 2 alpha from the subgenome A minus the signal pep- tide
  • SEQ ID NO: 60 nucleotide sequence of the GhRca 2 alpha from the subgenome A minus the signal pep- tide
  • SEQ ID NO: 61 amino acid sequence of the GhRca 1 alpha from the subgenome D
  • SEQ ID NO: 62 nucleotide sequence of the GhRca 1 alpha from the subgenome D
  • SEQ ID NO: 63 amino acid sequence of the GhRca 1 alpha from the subgenome D minus the signal pep- tide
  • SEQ ID NO: 64 nucleotide sequence of the GhRca 1 alpha from the subgenome D minus the signal pep- tide
  • SEQ ID NO: 65 amino acid sequence of the GhRca 2 alpha from the subgenome D
  • SEQ ID NO: 66 nucleotide sequence of the GhRca 2 alpha from the subgenome D
  • SEQ ID NO: 67 amino acid sequence of the GhRca 2 alpha from the subgenome D minus the signal pep- tide
  • SEQ ID NO: 68 nucleotide sequence of the GhRca 2 alpha from the subgenome D minus the signal pep- tide
  • SEQ ID NO: 69 amino acid sequence of the GmRca 1 alpha
  • SEQ ID NO: 70 nucleotide sequence of the GmRca 1 alpha
  • SEQ ID NO: 71 amino acid sequence of the GmRca 1 alpha minus the signal peptide
  • SEQ ID NO: 72 nucleotide sequence of the GmRca 1 alpha minus the signal peptide
  • SEQ ID NO: 73 amino acid sequence of the GmRca 2 alpha
  • SEQ ID NO: 74 nucleotide sequence of the GmRca 2 alpha
  • SEQ ID NO: 75 amino acid sequence of the GmRca 2 alpha minus the signal peptide
  • SEQ ID NO: 76 nucleotide sequence of the GmRca 2 alpha minus the signal peptide
  • SEQ ID NO: 77 amino acid sequence of the GmRca 3 alpha
  • SEQ ID NO: 78 nucleotide sequence of the GmRca 3 alpha
  • SEQ ID NO: 79 amino acid sequence of the GmRca 3 alpha minus the signal peptide
  • SEQ ID NO: 80 nucleotide sequence of the GmRca 3 alpha minus the signal peptide
  • SEQ ID NO: 81 amino acid sequence of the ZmRca alpha
  • SEQ ID NO: 82 nucleotide sequence of the ZmRca alpha
  • SEQ ID NO: 83 amino acid sequence of the ZmRca alpha minus the signal peptide
  • SEQ ID NO: 84 nucleotide sequence of the ZmRca alpha minus the signal peptide
  • SEQ ID NO: 85 amino acid sequence of the AtRca alpha
  • SEQ ID NO: 86 nucleotide sequence of the AtRca alpha
  • SEQ ID NO: 87 amino acid sequence of the AtRca alpha minus the signal peptide
  • SEQ ID NO: 88 nucleotide sequence of the AtRca alpha minus the signal peptide
  • SEQ ID NO: 89 amino acid sequence of the TaRca 2 beta from the subgenome A
  • SEQ ID NO: 90 amino acid sequence of the TaRca 2 beta from the subgenome B
  • SEQ ID NO: 91 amino acid sequence of the TaRca 2 beta from the subgenome D
  • SEQ ID NO: 92 nucleotide sequence of the T-DNA PubiZm: :hpRca
  • SEQ ID NO: 93 nucleotide sequence of the guide RNA gl
  • SEQ ID NO: 94 nucleotide sequence of the guide RNA g2
  • SEQ ID NO: 95 nucleotide sequence of the guide RNA g 13
  • SEQ ID NO: 96 nucleotide sequence of the guide RNA g9
  • SEQ ID NO: 97 nucleotide sequence of the T-DNA 35S-cab22L::AtRca-WT
  • SEQ ID NO: 98 nucleotide sequence of the T-DNA 35S-cab22L::AtRca-K-to-R
  • SEQ ID NO: 99 nucleotide sequence of the TaRca-WT
  • SEQ ID NO: 100 nucleotide sequence of the TaRca-K-to-R
  • SEQ ID NO: 101 nucleotide sequence of the target sequence in RCA 2
  • SEQ ID NO: 102 nucleotide sequence of the forward primer
  • SEQ ID NO: 103 nucleotide sequence of the reverse primer
  • SEQ ID NO: 104 nucleotide sequence of the reference probe
  • SEQ ID NO: 105 nucleotide sequence of the HT-19-020 forward primer
  • SEQ ID NO: 106 nucleotide sequence of the HT-19-020 reverse primer
  • SEQ ID NO: 107 nucleotide sequence of the NPTII forward primer
  • SEQ ID NO: 108 nucleotide sequence of the NPTII reverse primer
  • SEQ ID NO: 109 nucleotide sequence of the NPTII probe target
  • SEQ ID NO: 110 nucleotide sequence of the bar forward primer
  • SEQ ID NO: 111 nucleotide sequence of the bar reverse primer
  • SEQ ID NO: 112 nucleotide sequence of the bar probe target
  • Example 1 material and methods for the in vitro determination of the ADP inhibition of Rea proteins
  • Rca protein concentration was deter- mined using Protein Assay Dye Rcagent Concentrate (Bio-Rad, Hercules, CA, USA) with a bovine serum albumin (BSA) standard and molar concentration calculated using the molecular masses of 50,954 and 47, 110 Da for the an ad b isoforms, respectively.
  • BSA bovine serum albumin
  • Rubisco was extracted from the leaves of Triticum es ativum CV. Fielder (wheat) grown in a greenhouse under standard physiological conditions. Leaves were harvested greater than four hours into the light period and immediately frozen in liquid N and stored at -80°C until extraction. Frozen leaf tissue was ground into a fine powder using liquid N and a mortar and pestle. While on ice, leaf powder was added to and repeatedly vortexed in an extraction buffer consisting of 100 mM Tris pH 8.0, 1 mM EDTA, 10 mM MgCl2, 2 mM DTT, 2% W/V PVPP and protease inhibitor cocktail, before being passed through a single layer of Miricloth and Lingette Gaze to remove solid matter.
  • an extraction buffer consisting of 100 mM Tris pH 8.0, 1 mM EDTA, 10 mM MgCl2, 2 mM DTT, 2% W/V PVPP and protease inhibitor cocktail, before being passed through a single layer of Miricloth and
  • the sample was spun at 24,000 g for 20-min at 4°C and supernatant kept. 35% V V of saturated ammonium sulfate was added and the sample kept on ice for 30-min before re-spinning. To the supernatant 60% V/V of saturated ammonium sulfate was added dropwise and slowly stirred at 4°C for 30 min before being re-spun. The resulting pellet was suspended in a sample buffer of 100 mM Tricine pH 8.0, 0.5 mM EDTA and desalted into the same buffer using PD-10 desalting columns. 20% glycerol was added and the sample aliquoted into 50 pi volumes before being snap frozen and stored at -80°C until use. Final purity of Rca and Rubisco were high.
  • the assay was scaled down to 100 ml reactions and measured in Coster 96-well flat-bottom polystyrene plates (Coming, NY, USA), heated to 25°C using an Eppendorf Thermomixer (Eppendorf, Hamburg, Germany).
  • reaction solution with final volume of 80 ml was added consisting of N 2 sparged MiliQ H 2 O, 5% W V PEG-4000, 100 mM Tricine pH 8, 10 mM MgCl 2 , 10 mM NaHCCfi, 5 mM DTT, 2.4 U ml Enolase, 3.75 U ml Phosphoenolpyruvate carboxylase, 6 U ml Malate dehydrogen- ase, 0.2 mM 2,3-bis-Phosphoglycerate, 4 U ml d-2, 3 -phosphogly cerate mutase, 10 U ml carbonic anhydrase and 0.8 mM NADH.
  • ATP regenerating system consisting of 4 mM phosphocreatine and 20 U ml creatine phosphokinase was added. ATP and ADP were added at concentrations indicated in the text.
  • a final volume of 20 ml consisted of 0.25 ⁇ 0.05 mM of Rubisco active sites (see below for Rubisco active site quantification) added to either; 1) an activation solution (N2 sparged MiliQ H 2 O, 20 mM Tricine pH 8, 20 mM NaHCO 3 and 10 mM MgCL 2 ) to determine Rubisco total carbamylated activity (ECM), or 2) 4 mM of Ribulose-l,5-bisphosphate (RuBP; 99% pure) for Rubisco substrate inhibi- tion (ER).
  • the quantification of ECM regenerated reactions by Rca per minute was calculated by the method outlined by Loganathan et al. 2016 over the first 4-min period of measurements .
  • the amount of Rubisco active sites added to the assay was determined from the slope of a linear regression through the data points correspond- ing to the first 60-sec of 3-Phosphoglycetic acid (3PG) product generated from ECM samples and factoring in a wheat Rubisco reaction rate constant (K cat ) of 2.1 at 25°C (Hermida-Carrera et al., 2016, Response in Crops. Plant Physiol. 171, 2549-2561).
  • the amino-acid position 428 for the wheat TaRca2-a isoform and 432 for the wheat TaRcal- b isoform were mutated from a native lysine (K) to either an arginine (R) or glutamine (Q).
  • the mutation to an arginine mimics a lysine residue in a deacetylated state while the mutation to a glutamine mimics a lysine residue in an acetylated state.
  • the K428Q mutant had a significantly slower and K428R faster V max than the TaRca2-a wild type (Fig. 3A, Table 2).
  • K428Q was intermediate between the TaRca2-a wild type and K428R variant.
  • the K428Q mutant Rca results in a reduced activity and increased ATP affinity of the Rubisco enzyme compared to a corresponding wild type Rca
  • the K428R mutant Rca results in an increased activity and increased ATP affinity of the Rubisco enzyme compared to a corresponding wild type Rca.
  • the K428R mutant Rca results in an increased ATP affinity of the Rubisco enzyme compared to the K428Q Rca mutant.
  • a comparison of the wheat b isoform variants of Rca shows that despite the TaRca2-b isoform of wheat being sensitive to ADP inhibition it was less so than the isof aorm spliced variant, evident in it having a significantly lower K half than TaRca2-a (Fig. 3B, Table 2).
  • the TaRca1-b variant coded by a separate gene and with substantial genetic differences from the Rca2 spliced variants, had even more affinity for ATP with a K half significantly less than both Rca2 and Rc aa2 b isoforms and not significantly different from the TaRca2-a K428R mutant.
  • TaRca1-b had a significantly slower V max than the TaRca2 spliced variants.
  • Example 3 Generation of wheat plants with reduced level of endogenous Rca a proteins
  • the constitutive promoter region of the Ubiqui- tin gene of Zea mays according to the sequence from nucleotide position 157 to 2153 of SEQ ID NO: 92, the hairpin DNA fragment targeting the Rca genes from the subgenomes A, B and D of wheat according to the sequence from nucleotide position 2162 to 3543 of SEQ ID NO: 92, and the 3 ' untranslated sequence of the 35 S transcript gene of Cauliflower mosaic vims according to the sequence from nucleotide position 3547 to 3771 of SEQ ID NO: 92 were assembled in a vector which contains the bar selectable marker cassette (position 3856 to 5520 of SEQ ID NO: 92) to result in the T-DNA PubiZm::hpRca (SEQ ID NO: 92).
  • the recombinant vector comprising the expression cassettes PubiZm: :hpRca is used to stably transform wheat using the method described in Yuji Ishida et al. 2015, Methods in Molecular Bi- ology, 1223: 189-198. Homozygous and null segregant plants are selected.
  • a mutagenized wheat population was constructed by EMS mutagenesis. Based on sequencing of the region around the Rca genes, mutant plants with a knock-out mutation in the Rca gene from either the B subgenome, from the A subgenome or from the D subgenome are identified. The ho- mozygous mutant plants and their wildtype segregants are retrieved.
  • Such mutant plants are crossed to produce double mutant plants with a knock-out mutation in the Rca gene from both the subgenome A and B, or from both the subgenome A and D or from both the subgenome B and D.
  • Such resulting double mutant plants are further crossed to produce mutant plants with a knock-out mutation in the Rca gene from all three subgenomes (namely A, B and D).
  • Guide RNAs for CRISPR-mediated gene editing targeting the mRNA coding sequence pref- erably the protein coding sequence of the Rca gene from the D subgenome, targeting the mRNA coding sequence, preferably the protein coding sequence of the Rca genes from both the D and the A subgenomes, targeting the mRNA coding sequence, preferably the protein coding sequence of the Rca genes from both the A and the B subgenomes, or targeting the mRNA coding sequence, preferably the protein coding sequence of the Rca genes from both the A, the B and the D subge- nomes were designed by using e.g. the CAS-fmder tool.
  • the guide RNAs were tested for targeting efficiency by PEG-mediated transient co-delivery of the gRNA expression vector with an expres- sion vector for the respective nuclease, e.g. Cas9 or Cpfl, under control of appropriate promoters, to protoplasts of a wheat line containing the Rca genes. Genomic DNA was extracted from the protoplasts after delivery of the guide RNA and nuclease vectors. After PCR amplification, integ- rity of the targeted Rca gene sequence was assessed by sequencing.
  • the most efficient guide RNAs were used for stable gene editing in wheat.
  • the selected guide RNAs are gl (SEQ ID NO: 93) targeting the subgenomes A, B and D; g2 (SEQ ID NO: 94) targeting the subgenomes A and D; g 13 (SEQ ID NO: 95) targeting the subgenomes A and B; and g9 (SEQ ID NO: 96) targeting the subgenome D.
  • the selected guide RNA expres- sion vector, together with a nuclease expression module and a selectable marker gene were intro- cuted into wheat embryos using e.g. particle gun bombardment.
  • Transgenic plants showing re- sistance to the selection agent were regenerated using methods known to those skilled in the art. At least 12 transgenic TO plants containing gene targeting events, preferably small deletions or inser- tions resulting in a non-functional Rca gene were identified by PCR amplification and sequencing. Examples of knock-out mutant plants obtained are shown is table 3.
  • version 1 seedlot homozygous for the 4A03 mutation and a corresponding wild-type seed- lot
  • b. version 2 seedlot homozygous for the 4B03 mutation and a corresponding wild-type seed- lot
  • c. version 3 seedlot homozygous for the 4A02 and 4D03 mutation and a corresponding wild- type seedlot
  • d. version 4 seedlot homozygous for the 4B04 and 4D02 mutation and a corresponding wild- type seedlot.
  • Immature embryos 2-3 mm size, were isolated from sterilized ears of wheat cv. Fielder and bombarded using the PDS-1000/He particle delivery system. Plasmid DNA of the the ABE7.10 and the gRNA vectors were mixed with the plasmid DNA pIB26. The vector pIB26 contains an cgfp-bar fusion gene. Bombarded immature embryos were moved to PPT containing selection media, and PPT resistant calli were selected and transferred to regeneration media for shoot for- mation.
  • a ddPCR as- say was performed using the primer pair (Forward 5’: CTCATCAGATCGTCCAAAACAA, SEQ ID NO: 102), Reverse 5’: TCGAAGTTCTTGGCAGTCT, SEQ ID NO: 103) and the reference probe 5 ⁇ EC- CACAGCAAGGTACTTTGCCTGT, SEQ ID NO: 104) to detect the presence of any RCA2 homoeallele and the edit probe 5’ FAM-CC+T+C+TA+C+C+TG or the edit probe 5’ FAM-CC+C+C+CTACCTG to amplify RCA2 homoeoalleles with the desired edit AAA >AGA or AAA > AGG, respectively.
  • The’+’ preceeding the nucleotide refers to the presence of an LNA base (locked nucleic acid base).
  • each plantlet pool is derived from a single immature embryo
  • all plantlets derived from a single immature embryo are considered as an independent edited event, although we can’t exclude that there might be multiple independent edited events between individual shoots derived from a single immature embryo scored as positive in the 2 nd dd PCR.
  • edits could be detected by dd PCR at a frequency between l . l%-20.2%.
  • individual shoots with an edit frequency of ⁇ 10 to 20% were observed indicating that one out of the 6 alleles might be precisely edited.

Abstract

The present invention relates to the field of agriculture. In particular, the invention provides a Rca protein with reduced ADP inhibition, a recombinant gene, plants comprising the recombinant gene and a method to improve yield of a plant.

Description

RUBISCO ACTIVASE with reduced ADP inhibition and uses thereof
Field Of The Invention
[ 1 ] The present invention relates to methods and means to increase the ratio of Rubisco Activase (Rca) proteins having higher activity and reduced sensitivity to ADP inhibition in plants and increase the photosynthesis and yield of plants. In particular, the invention provides a Rca a protein having an increased protein activity and a reduced sensitivity to ADP inhibition compared to the native Rca a proteins.
Background
[2] Ribulose-l,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is the central enzyme of photosyn- thesis catalyzing the fixation of C02 into sugars (Andersson, 2008, J. Exp. Bot. 59, 1555-1568). Rubisco is susceptible to inhibition by catalytic misfire products, in a process sometimes referred to as failover ( Pearce, F. G., and Andrews, T. J., 2003, J. Biol. Chem. 278, 32526-32536; Pearce, F. G., 2006, Biochem. J. 399, 525-534; Schrader et al., 2006, Funct. Plant Biol. 33, 921-929). Indeed the sugar substrate of Rubisco, Ribulose-l,5-bisphosophate (RuBP) is an inhibitor of Ru- bisco (often referred to as the ER complex) if the active site is not initially primed by the binding of a Mg2+ ion and carbamylated with a molecule of C02 separate from the one catalyzed (often referred to as the ECM complex) (Jordan, D. B., and Chollet, R., 1983, J. Biol. Chem. 258, 13752- 13758; Cleland et al., 1998, Chem. Rev. 98, 549-562). The removal of inhibitors from the Rubisco active site and hence regulation of Rubisco and ultimately photosynthesis is in large part due to its chaperone partner-protein, Rubisco activase (Rca) (Portis, A. R., 2003, Photosynth. Res. 75, 11- 27; Salvucci, M. E., and Anderson, J. C., 1987, Plant Physiol. 85, 66-71). Rca is a member of the AAA+ superfamily of enzymes, all of which bind ATP nucleotide as a co-factor and many of which are chaperones and involved in maintaining function of partner proteins (Erzberger, J. P., and Ber- ger, J. M., 2006, Annu. Rev. Biophys. Biomol. Struct. 35, 93-114). Rca clears tightly bound sugar substrates and inhibitors from the active site of Rubisco through a mechanism which is not clearly characterized for higher plants (Mueller-Cajar et al., 2014, Photosynth. Res. 119, 191-201). Diffi- culty in characterizing interaction with Rubisco arises as the active multimeric Rca complex is dynamic and altered by co-factors such as nucleotides and Mg2+, as well as concentration and solvent dependent self-association (Henderson et al., 2013, Spec. Sect. Chem. Mech. Phosphatases Diesterases Triesterases. 1834, 87-97; Keown, J. R., and Pearce, F. G., 2014, Biochem. J. 464, 413; Wang et al., 2017, bioRxiv. 10.1101/191742; Wang et al., 1993, Biochim. Biophys. Acta BBA - Protein Struct. Mol. Enzymol. 1202, 47-55; Salvucci, M. E., 1992, Arch. Biochem. Biophys. 298, 688-696). Structural and mutational analysis suggests that the active holoenzyme consists of a hexamer (Stotz et al., 2011, Nat Struct Mol Biol. 18, 1366-1370), yet activity might occur for smaller and larger complexes (Keown, J. R., and Pearce, F. G., 2014, Biochem. J. 464, 413; Wang, et al., 1993, Biochim. Biophys. Acta BBA - Protein Struct. Mol. Enzymol. 1202, 47-55). One certainty is that ATP is required by Rca to regenerate Rubisco active sites and ADP inhibits the activity of Rca (Robinson, S. P., and Portis Jr, A. R., 1989, Arch. Biochem. Biophys. 268, 93-99). This provides a tight control over photosynthesis as during high irradiance ADP/ATP ratios are low and Rca will enable Rubisco to maintain activity, while at low irradiance (for example in cloudy weather) ADP/ATP ratios are high and Rubisco activity will decline as a lack of Rca activity will lead to progressively more active sites of Rubisco becoming impaired.
[3] In most higher plant species studied so far there are two isoforms of Rca due to either alternative splicing of pre-mRNA from a single gene, or encoding by two separate genes (Nagarajan, R., and Gill, K. S., 2017, Plant Mol. Biol. 10.1007/ s 1 1 103-017-0680-y). The longer polypeptide is referred to as the a-isoform and the shorter the b-isoform. The primary structural difference between the a and b-isoforms is 25 to 45 additional residues attached to the C-terminal of the former. A conserved characteristic of this C-terminal extension (CTE) is the presence of two cysteine residues which form a disulfide bond under non-reduced conditions and requires specific reduction by the redox regulated chloroplast protein thioredoxin-f (Zhang, N., and Portis, A. R., 1999, Proc. Natl. Acad. Sci. 96, 9438-9443). Thioredoxin-f actively reduces disulfide bonds in target proteins during high irradiance. Reduction of the C-terminal disulfide bond leads to a decrease in ADP inhibition and increased ATP affinity of the Rca a isoform in Arabidopsis (Zhang, N., and Portis, A. R., 1999, Proc. Natl. Acad. Sci. 96, 9438-9443; Zhang et al., 2001, Photosynth. Res. 68, 29-37). Thus the enhancement of a-isoform disulfide bond reduction during high irradiance by thioredoxin-f reduces ADP inhibition, promotes ATP affinity and thus provides a secondary mechanism for coordinating the regulation of Rubisco to light conditions. In some species that express both an a and b-isoform such as Arabidopsis ( Arabidopsis thaliana) the a- isoform is sensitive to ADP inhibition while the b-isoform has relatively minimal sensitivity, while other species such as tobacco ( Nicotiana tabacum) only expresses a b-isoform that is sensitive to ADP (Carmo-Silva, A. E., and Salvucci, M. E., 2013, Plant Physiol. 161, 1645-1655). Irrespective of the sensitivity of the b-isoform it seems that due to the indiscriminate incorporation of both isoforms into the active heteroligomeric Rca complex, changes in ADP inhibition for the a isoform translate to inhibition characteristics for the final active holoenzyme (Zhang et al., 2001, Photosynth. Res. 68, 29-37; Carmo-Silva, A. E., and Salvucci, M. E., 2013, Plant Physiol. 161, 1645-1655). Little is known about the ADP inhibition of RCA from monocot species. Rice and wheat express both an a and b-isoform, with wheat expressing an a-isoform (TaRca2-a) and two variants of the b-isoform, the spliced variant of the a, referred to as TaRca2-b and a unique b- isoform encoded by a separate gene, referred to as TaRcal-b ( To et al., 1999, Planta. 209, 66- 76; Carmo-Silva et al., 2015, Plant Cell Environ. 38, 1817-1832). The sensitivity of these wheat isoforms to ADP and ATP is not known.
[4] There remains thus a need to improve the activity of the Rca proteins via a reduction of its sensitivity to ADP inhibition.
Summary
[5] In one aspect, the invention provides a Rca a protein (Rubisco activase) comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7. The Rca a protein may comprise an amino acid sequence selected from (a) the amino acid sequence of SEQ ID NOs: 3, 7; 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87, wherein the amino acid at a position corresponding to position 382 of SEQ ID NO: 7 is substituted with an arginine (b) an amino acid sequence having at least 90% identity to the amino acid sequences of SEQ ID NOs: 3, 7; 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87 and comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7, (c) the amino acid sequences of SEQ ID NOs: 3, 7; 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87, wherein the amino acid at a position corresponding to position 382 of SEQ ID NO: 7 is substituted with an arginine and further comprising a chloroplast targeting peptide, or (d) an amino acid sequence having at least 90% identity to the amino acid sequences of SEQ ID NOs: 3, 7; 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87, and comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7, further comprising a chloroplast targeting peptide, (e) the amino acid sequence of SEQ ID NOs: 1, 5; 9, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 or 85, wherein the amino acid at a position corresponding to position 382 of SEQ ID NO: 7 is substituted with an arginine, and (f) an amino acid sequence having at least 90% identity to the amino acid sequences of SEQ ID NOs: 1, 5; 9, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 or 85 and comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7. This Rca a protein may have an increased maximal Rubisco activase activity and/or a reduced ADP inhibition of its Rubisco activase activity compared to an Rca 2a protein not comprising said arginine at a position corresponding to position 382 of SEQ ID NO: 7.
[6] A nucleic acid encoding the Rca a protein according to the invention is also provided and it may comprise a coding nucleotide sequence selected from (a) the nucleotide sequence of SEQ ID Nos: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88, wherein the codon encoding the amino acid at a position corresponding to position 382 of SEQ ID NO: 7 is substituted with an codon encoding an arginine, or the complement thereof, and (b) a nucleotide sequence having at least 60% identity with the nucleotide sequence of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88 and encoding the Rca a protein according to the invention, or the complement thereof. In yet another embodiment, an allele of a Rca a gene encoding the protein according to the invention is also provided.
[7] In another embodiment, a gene, such as a recombinant gene, is provided which comprises the following operably linked elements (a) a promoter, preferably expressible in plants, (b) a nucleic acid encoding the Rca a protein according to the invention and, optionally (c) a transcription ter- mination and polyadenylation region, preferably a transcription termination and polyadenylation region functional in plants. Said promoter may be a constitutive promoter, tissue-specific promoter or an inducible promoter and said nucleic acid encoding the Rca a protein may comprise the coding nucleotide sequence according to the invention.
[8] In yet another embodiment, a cell is provided which comprises the protein of the invention, the nucleic acid of the invention or at least one Rca a allele according to the invention. The plant cell may further comprise (a) at least one knock-out allele of a Rca gene, or (b) a recombinant gene capable of suppressing specifically the expression of the endogenous Rca genes. A plant, plant part or seed consisting essentially of the plant cells of the invention are also provided.
[9] In another aspect, a method of increasing the maximal Rubisco activase activity and/or of reducing the ADP inhibition of the Rubisco activase activity of a Rca a protein is provided which comprises introducing an amino acid substitution to the amino acid sequence of said Rca a protein, wherein the amino acid substitution is a substitution of the amino acid at a position corresponding to position 382 of SEQ ID NO: 7 with an arginine. The maximal Rubisco activase activity may be increased by about 10% and the ADP inhibition of the Rubisco activase activity may be reduced by about 10%.
[10] In yet another aspect, a method for increasing the ratio of an arginine Rca a protein variant in plants comprising (a) providing to cells of a plant the recombinant gene provided herein, and re- ducing the expression of endogenous Rca protein in said plant cells, wherein said ratio is increased compared to a control plant cell not comprising said recombinant gene; or (b) introducing into cells of a plant at least one Rca a allele according to the invention, wherein said ratio is increased com- pared to a control plant cell not comprising said Rca a allele. The expression of endogenous Rca a protein may be reduced by (a) introducing into said cells of a plant at least one knock-out mutant Rca allele; or (b) providing said cells of a plant with a second recombinant gene capable of sup- pressing specifically the expression of the endogenous Rca genes.
[1 1] A method for increasing photosynthetic activity and/or increasing yield of a plant is also pro- vided which comprises (a) increasing the ratio of an arginine Rca a protein variant, and (b) regen- erating said plant, wherein said photosynthetic activity and/or yield is increased compared to a plant not comprising said increased ratio of an arginine Rca a protein variant. The yield may be seed yield or thousand seed weight.
[12] A method for producing a plant with an increased photosynthetic activity and /or with an in- creased yield is provided. Said method comprises (a) increasing the ratio of an arginine Rca a protein variant according to the invention in a plant and (b) regenerating said plant, wherein said photosynthetic activity and/or said yield is increased compared to a plant not comprising said in- creased ratio of an arginine Rca a protein variant.
[13] A method for producing a plant with an increased photosynthetic activity and/or increased yield is also provided which comprises (a) increasing the ratio of an arginine Rca a protein variant; and (b) regenerating said plant, wherein said photosynthetic activity and/or yield is increased com- pared to a plant not comprising said increased ratio of an arginine Rca a protein variant.
[14] Also provided is the use of the arginine Rca a protein variant according to the invention, the nucleic acid encoding an arginine Rca a protein variant according to the invention, the recombinant gene according to the invention or the arginine Rca a allele according to the invention, to increase the ratio of an arginine Rca a protein variant in plant, to increase photosynthetic activity of a plant, to increase yield of a plant or to produce a plant with increased photosynthetic activity or with an increased yield.
Brief Description Of The Drawings
[15] Figure 1. ADP inhibition of Rca activation velocity for wheat (TaRca) and rice (OsRca) a and b isoforms. Rca velocity in the presence of 5 mM ATP substrate and varying concentrations of ADP inhibitor was normalized to initial velocity in the absence of inhibitor (Vi/V0) and plotted against the fraction of ADP to ATP. Values are the means ± SD of 3 experimental replicates.
[16] Figure 2. C-terminal alignment of wheat (TaRca) and Arabidopsis (AtRca) a and b Rca isoforms indicating the mutation made in this study. The amino-acid position 428 for the wheat TaRca2-a isoform and 432 for the wheat TaRca1-b isoform were mutated from a native lysine (K) to either an arginine (R) or glutamine (Q).
[17] Figure 3. ATP substrate dependent enzyme kinetic curves of the TaRca2-a isoform and de- rived CTE mutant (A), and wheat b isoforms and TaRca1-b derived mutant (B). The enzymatic velocity of Rubisco reactivation by Rca (ECM regenerated reactions per minute per Rca monomer) was plotted against ATP concentration added to assays. An ATP regenerating system using phos- phocreatine and creatine phosphokinase was used to limit ADP buildup due to ATP hydrolysis by Rca. Curves are Hill equations [ =Vmax*S*h/(Khalf* Sh ) ] with Vmax the maximum velocity reached, Khalf the ATP substrate corresponding to half maximal velocity in mM, h is the Hills-slope constant and S the ATP substrate concentration in mM. The Vmax, Khalf and h values were generated from iterative fits using a least-squares model and values for each curve are presented in Table 2. Values are the means ± SD of 3-5 experimental replicates.
[18] Figure 4. Inhibitor dose-response curves of TaRca2-a and C-terminal extension mutant (A), and for the wheat b isoforms and TaRca1-b mutant (B). The relationship between IC50 and Khalf for the a variants (C) and b variants (D). (A, B) Rca velocity in the presence of varying concentrations of ADP inhibitor (I) was modeled by ordinary least-squares dose-response curves using the follow- ing equation [Vi/V0= Vi/V0min + (Vi/V0max - Vi/V0min)/1+10I-LogIC50], with the IC50 value correspond- ing to the ADP inhibitor concentration in mM at which half inhibitor free velocity is reached. The log transformed ADP concentrations added to assays are given on the x-axis, while 500 mM of ATP substrate was added in all assays. Calculated IC50 values are presented in Table 2. (C, D) Scatter- plots of IC50 and Khalf for the Rca variants and fit with a linear regression model in the case of the a variants. Values are the means ± SD of 3-5 experimental replicates.
[ 19] Figure 5. ATP substrate dependent enzyme kinetic curves of TaRca2-a (A) and K428R mutant (B) with varying concentrations of ADP inhibitor (I) added as indicated on the right of individual curves. A competitive-inhibition model fitted the data best (global R2>0.98 for both variants) using the equations [Khalf -Obs= Khalf h( 1 +I/Ki) ] and [ V=Vmax* Sh/(Khalf -Obs+.Sh) ]: where Khalf is the ATP sub- strate corresponding to half maximal velocity in mM, h is the Hills-slope, S is the ATP substrate concentration and 7 is the ADP inhibitor concentration in mM. The apparent inhibition binding constant (K,) was determined to be 4.9±1.5 mM for the TaRca2-a and 3.4±1.4 mM for K428R through iteration of the curves.
Detailed Description
[20] The present invention is based on the surprising discovery that substituting the amino acid at the position corresponding to position 382 of SEQ ID NO: 7 with an arginine reduces the ADP inhibition of the Rca a protein activity and increases the maximal Rubisco activase ( Rca) activity of the Rca a protein.
Definitions
[21] As used herein for protein sequences, the term "percent sequence identity" refers to the per- centage of conserved amino acids between two segments of a window of optimally aligned poly- peptides. Optimal alignment of sequences for aligning a comparison window are well-known to those skilled in the art and the percentage of conservation may be calculated by matrix such as BLOSUM (Blocks Substitution Matrix) and PAM (Point Accepted Mutation) (Henikoff and Heni- koff, 1992, PNAS 89(22): 10915-10919). An "identity fraction" for aligned segments of a test se- quence and a reference sequence is the number of identical or conserved components that are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction times 100. The comparison of one or more protein sequences may be to a full-length protein sequence or a portion thereof, or to a longer protein sequence.
[22] The term "protein" interchangeably used with the term“polypeptide” as used herein describes a group of molecules consisting of more than 30 amino acids, whereas the term“peptide” describes molecules consisting of up to 30 amino acids. Proteins and peptides may further form dimers, tri- mers and higher oligomers, i.e. consisting of more than one (poly)peptide molecule. Protein or peptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corre- sponding higher order structures are, consequently, termed homo- or heterodimers, homo- or het- erotrimers etc. The terms "protein" and“peptide” also refer to naturally modified proteins or pep- tides wherein the modification is effected e.g. by glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.
[23] The term "variant" with respect to the nucleotide sequences of the invention is intended to mean substantially similar sequences. Naturally occurring allelic variants such as these can be iden- tified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as herein outlined before. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis of any one of SEQ ID NOs: 1, 5; 9, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 or 85. Generally, nucleotide sequence variants of the invention will have at least 40%, at least 50%, at least 60%, to at least 70%, e.g., preferably at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, to at least 79%, generally at least 80%, e.g., at least 81% to at least 84%, at least at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, to at least 98% and at least 99% nucle- otide sequence identity to the native (wild type or endogenous) nucleotide sequence. Derivatives of the DNA molecules disclosed herein may include, but are not limited to, deletions of sequence, single or multiple point mutations, alterations at a particular restriction enzyme site, addition of functional elements, or other means of molecular modification. Techniques for obtaining such de- rivatives are well-known in the art (see, for example, J. F. Sambrook, D. W. Russell, and N. Irwin (2000) Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3. Cold Spring Harbor Laboratory Press). Those of skill in the art are familiar with the standard resource materials that describe specific conditions and procedures for the construction, manipulation, and isolation of macromolecules (e.g., DNA molecules, plasmids, etc.), as well as the generation of recombinant organisms and the screening and isolation of DNA molecules. [24] As used herein for nucleotide sequences, the term "percent sequence identity" refers to the percentage of identical nucleotides between two segments of a window of optimally aligned DNA. Optimal alignment of sequences for aligning a comparison window are well-known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Wa- terman (Waterman, M. S. Introduction to Computational Biology: Maps, sequences and genomes. Chapman & Hall. London (1995), the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol., 48:443-453 (1970), the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci., 85:2444 (1988), and preferably by computerized implementations of these algo- rithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG (Registered Trade Mark), Wisconsin Package (Registered Trade Mark from Accelrys Inc., San Diego, Calif.). An "identity fraction" for aligned segments of a test sequence and a reference sequence is the num- ber of identical components that are shared by the two aligned sequences divided by the total num- ber of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the iden- tity fraction times 100. The comparison of one or more DNA sequences may be to a full-length DNA sequence or a portion thereof, or to a longer DNA sequence.
[25] The term "recombinant gene" refers to any artificial gene that contains: a) DNA sequences, including regulatory and coding sequences that are not found together in nature, or b) sequences encoding parts of proteins not naturally adjoined, or c) parts of promoters that are not naturally adjoined. Accordingly, a recombinant gene may comprise regulatory sequences and coding se- quences that are derived from different sources, i.e. heterologous sequences, or comprise regulatory sequences, and coding sequences derived from the same source, but arranged in a manner different from that found in nature.
[26] The term "heterologous" refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources. For example, a promoter is heterologous with respect to an operably linked DNA region, such as a coding sequence if such a combination is not normally found in nature. In addition, a particular sequence may be "heterologous" with respect to a cell or organism into which it is inserted (i.e. does not naturally occur in that particular cell or organism). For example, the recombinant gene disclosed herein is a heterologous nucleic acid.
[27] The term“endogenous” relates to what originate from within the plant or cell. An endogenous gene is thus a gene originally found in a given plant or cell.
[28] “Isolated nucleic acid”, used interchangeably with“isolated DNA” as used herein refers to a nucleic acid not occurring in its natural genomic context, irrespective of its length and sequence. Isolated DNA can, for example, refer to DNA which is physically separated from the genomic context, such as a fragment of genomic DNA. Isolated DNA can also be an artificially produced DNA, such as a chemically synthesized DNA, or such as DNA produced via amplification reac- tions, such as polymerase chain reaction (PCR) well-known in the art. Isolated DNA can further refer to DNA present in a context of DNA in which it does not occur naturally. For example, iso- lated DNA can refer to a piece of DNA present in a plasmid. Further, the isolated DNA can refer to a piece of DNA present in another chromosomal context than the context in which it occurs naturally, such as for example at another position in the genome than the natural position, in the genome of another species than the species in which it occurs naturally, or in an artificial chromo- some.
[29] Hybridization occurs when the two nucleic acid molecules anneal to one another under appro- priate conditions. Nucleic acid hybridization is a technique well known to those of skill in the art of DNA manipulation. The hybridization property of a given pair of nucleic acids is an indication of their similarity or identity. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a com- plex mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mis- matches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence. "Stringent hybridization condi- tions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence-dependent, and are differ- ent under different environmental parameters. An example of highly stringent wash conditions is 0.15 M NaCI at 72°C for about 15 minutes. An example of stringent wash conditions is a 0.2 X SSC wash at 65°C for 15 minutes. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2 X (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This oc- curs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
[30] The phrases“DNA”, "DNA sequence," "nucleic acid sequence," "nucleic acid molecule"“nu- cleotide sequence” and“nucleic acid” refer to a physical structure comprising an orderly arrange- ment of nucleotides. The terms“sequence” and“molecule” may be used interchangeably. The DNA sequence or nucleotide sequence may be contained within a larger nucleotide molecule, vector, or the like. In addition, the orderly arrangement of nucleic acids in these sequences may be depicted in the form of a sequence listing, figure, table, electronic medium, or the like.
[31] As used herein“comprising” is to be interpreted as specifying the presence of the stated fea- tures, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A recombinant gene comprising a nucleic acid which is functionally or structurally defined, may comprise additional DNA regions etc. However, in context with the present disclosure, the term “comprising” also includes“consisting of’.
[32] It is understood that when referring to a word in the singular (e.g. plant or allele), the plural is also included herein (e.g. a plurality of plants, a plurality of alleles). Thus, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the ele- ment is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
[33] As used herein, the term“allele(s)” means any of one or more alternative forms of a gene at a particular locus. In a diploid (or amphidiploid) cell of an organism, alleles of a given gene are located at a specific location or locus (loci plural) on a chromosome. One allele is present on each chromosome of the pair of homologous chromosomes.
[34] As used herein, the term“locus” (loci plural) means a specific place or places or a site on a chromosome where for example a gene or genetic marker is found. For example, the“ TaRca 2 A locus” refers to the position on a chromosome of the wheat A genome where a TaRca 2 A gene (and two TaRca 2 A alleles) may be found, while the“TaRca 2 B locus” refers to the position on a chromosome of the wheat B genome where a TaRca 2 B gene (and two TaRca 2B alleles) may be found and the“TaRca 2 D locus” refers to the position on a chromosome of the wheat D genome where a TaRca 2 D gene (and two TaRca 2 D alleles) may be found. Similarly, the“ OsRca locus” refers to the position on a chromosome of the rice genome where an Os Rca gene (and two Os Rca alleles) may be found, the“ BnRca 1 A locus” refers to the position on a chromosome of the Brassica napus A genome where a BnRca 1 A gene (and two BnRca 1 A alleles) may be found, the“ BnRca 2 A locus” refers to the position on a chromosome of the Brassica napus A genome where a BnRca 2 A gene (and two BnRca 2 A alleles) may be found, the“ BnRca 3 A locus” refers to the position on a chromosome of the Brassica napus A genome where a BnRca 3 A gene (and two BnRca 3 A alleles) may be found, the“ BnRca 1 C locus” refers to the position on a chromosome of the Brassica napus C genome where a BnRca 1 C gene (and two BnRca 1 C alleles) may be found, the“ BnRca 2 C locus” refers to the position on a chromosome of the Brassica napus C genome where a BnRca 2 C gene (and two BnRca 2 C alleles) may be found, the“ BnRca 3 C locus” refers to the position on a chromosome of the Brassica napus C genome where a BnRca 3 C gene (and two BnRca 3 C alleles) may be found, the“ GhRca 1 A locus” refers to the position on a chromosome of the Goss- ypium hirsutum A genome where a Gh Rca 1 A gene (and two Gh Rca 1 A alleles) may be found, the“Gh Rca 2 A locus” refers to the position on a chromosome of the Gossypium hirsutum A ge- nome where a GhRca 2 A gene (and two GhRca 2 A alleles) may be found, the“ GhRca 1 D locus” refers to the position on a chromosome of the Gossypium hirsutum D genome where a GhRca 1 D gene (and two GhRca 1 D alleles) may be found, the GhRca 2 D locus” refers to the position on a chromosome of the Gossypium hirsutum D genome where a GhRca 2 D gene (and two GhRca 2 D alleles) may be found, the“ GmRca 1 locus” refers to the position on a chromosome of the Glycine max genome where a GmRca 1 gene (and two GmRca 1 alleles) may be found, the“ GmRca 2 locus” refers to the position on a chromosome of the Glycine max genome where a GmRca 2 gene (and two GmRca 2 alleles) may be found, the“ GmRca 3 locus” refers to the position on a chromo- some of the Glycine max genome where a GmRca 3 gene (and two GmRca 3 alleles) may be found, the“ ZmRca locus” refers to the position on a chromosome of the maize genome where an ZmRca gene (and two ZmRca alleles) may be found, the AtRca locus” refers to the position on a chromo- some of the Arabidopsis thaliana genome where an AtRca gene (and two AtRca alleles) may be found.
[35] An Rca gene as used herein is a gene from which an iso aform of an Rca protein can be pro- duced. The same Rca gene may or may not also produce a b isoform of an Rca protein.
[36] “Wild type” (also written“wildtype” or“wild-type”), as used herein, refers to a typical form of a plant or a gene as it most commonly occurs in nature. A“wild type plant” refers to a plant with the most common phenotype of such plant in the natural population. A“wild type allele” refers to an allele of a gene required to produce the wild-type protein and wild type phenotype. By contrast, a“mutant plant” refers to a plant with a different rare phenotype of such plant in the natural popu- lation or produced by human intervention, e.g. by mutagenesis or gene editing, and a“mutant al- lele” refers to an allele of a gene required to produce the mutant protein and/or the mutant pheno- type.
[37] “Mutant” as used herein refers to a form of a plant or a gene which is different from such plant or gene in the natural population, and which is produced by human intervention, e.g. by mutagen- esis or gene editing , and a“mutant allele” refers to an allele which is not found in plants in the natural population or breeding population, but which is produced by human intervention such as mutagenesis or gene editing. [38] As used herein, the term“wild type allele” (e.g. wild type TaRca 2 B allele, wild type TaRca
2 A allele, wild type TaRca 2 D allele, wild type OsRca allele, wild type BnRca 1 A allele, wild type BnRca 2 A allele, wild type BnRca 3 A allele, wild type BnRca 1 C allele, wild type BnRca 2 C allele, wild type BnRca 3 C allele, wild type GhRca 1 A allele, wild type GhRca 2 A allele, wild type GhRca 1 D allele, wild type GhRca 2 D allele, wild type GmRca 1 allele, wild type GmRca 2 allele, wild type GmRca 3 allele, wild type ZmRca allele, or a wild type AtRca allele), means a naturally occurring allele found in plants, in particular wheat plants, rice plants, Brassica plants, soybean plants, cotton plants, maize plants or Arabidopsis plants, which encodes a functional a isoform Rca protein (e.g. a functional iso aform TaRca 2 B, isofo arm TaRca 2 A, a isoform TaRca 2D, a isoform OsRca, is aoform BnRca 1 A, a isoform BnRca 2 A, a isoform BnRca 3 A, a isoform BnRca 1 C, a isoform BnRca 2 C, is aoform BnRca 3 C, a isoform GhRca 1 A, a isoform GhRca 2 A, a isoform GhRca 1 D, a isoform GhRca 2 D, a isoform GmRca 1, i asoform GmRca 2, a isoform GmRca 3, a isoform ZmRca, or a isoform AtRca protein). In contrast, the term“mutant allele” (e.g. mutant TaRca 2 B allele, mutant TaRca 2 A allele, mutant TaRca 2 D allele, mutant OsRca allele, mutant BnRca 1 A allele, mutant BnRca 2 A allele, mutant BnRca 3 A allele, mutant BnRca 1 C allele, mutant BnRca 2 C allele, mutant BnRca 3 C allele, mutant GhRca 1 A allele, mutant GhRca 2 A allele, mutant GhRca 1 D allele, mutant GhRca 2 D allele, mutant GmRca 1 allele, mutant GmRca 2 allele, mutant GmRca 3 allele, mutant ZmRca allele, or mutant AtRca allele), as used herein, refers to an allele, which does not encode a functional isofor am Rca protein, i.e. an Rca allele encoding a non-functional iso aform Rca protein (e.g. a non-functional isofo arm TaRca 2 B, i asoform TaRca 2 A, a isoform TaRca 2 D, iso aform OsRca, isof aorm BnRca 1 A, a isoform BnRca 2 A, is aoform BnRca 3 A, a isoform BnRca 1 C, a isoform BnRca 2 C, isoform a BnRca 3 C, a isoform GhRca 1 A, a isoform GhRca 2 A, iso aform GhRca 1 D, a isoform GhRca 2 D, a isoform GmRca 1, is aoform GmRca 2, iso aform GmRca 3, isofo arm ZmRca, or isoform a AtRca protein) or an Rca allele encoding a functional isofo arm protein having an increase maximal Rca activity and/or a reduced ADP inhibition of its Rca activity compared to a wild type allele (e.g. a functional i asoform TaRca 2 B, is aoform TaRca 2 A, iso aform TaRca 2 D, isof aorm OsRca, a isoform BnRca 1 A, a isoform BnRca 2 A, isof aorm BnRca 3 A, a isoform BnRca 1 C, a isoform BnRca 2 C, a isoform BnRca 3 C, a isoform GhRca 1 A, a isoform GhRca 2 A, isof aorm GhRca 1 D, a isoform GhRca 2 D, a isoform GmRca 1, isofo arm GmRca 2, isofor am GmRca 3, isoform a ZmRca, or a isoform AtRca protein having an increase maximal Rca activity and/or a reduced ADP inhibition of its Rca activity compared to a wild type iso aform protein). A mutant Rca allele en- coding a non-functional a isoform Rca protein, as used herein, refers to an iso aform Rca protein having no biological activity or a significantly reduced biological activity as compared to the cor- responding wild-type functional i asoform Rca protein, or encoding no is aoform Rca protein at all. An Rca allele encoding a functional iso aform protein having an increase maximal Rca activity and/or a reduced ADP inhibition of its Rca activity compared to a wild type protein, as used herein, refers to a a isoform Rca protein variant as described below. A knock-out Rca allele is an equivalent term for a mutant Rca allele encoding a non-functional isof aorm Rca protein. A“382R” Rca allele is an equivalent term for an Rca allele encoding a functional isof aorm protein having an increase maximal Rca activity and/or a reduced ADP inhibition of its Rca activity compared to a wild type allele.
[39] “Mutagenesis”, as used herein, refers to the process in which plant cells (e.g., a plurality of cereal seeds or other parts, such as pollen, etc.) are subjected to a technique which induces muta- tions in the DNA of the cells, such as contact with a mutagenic agent, such as a chemical substance (such as ethylmethylsulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation (neutrons (such as in fast neutron mutagenesis, etc.), alpha rays, gamma rays (such as that supplied by a Cobalt 60 source), X-rays, UV-radiation, etc.), T-DNA insertion mutagenesis (Azpiroz-Leehan et al. (1997) Trends Genet 13: 152-156), transposon mutagenesis (McKenzie et al. (2002) Theor Appl Genet 105:23-33), or tissue culture mutagenesis (induction of somaclonal variations), or a combi- nation of two or more of these. Thus, the desired mutagenesis of one or more Rca 2 alleles may be accomplished by use of one of the above methods. While mutations created by irradiation are often large deletions or other gross lesions such as translocations or complex rearrangements, mutations created by chemical mutagens are often more discrete lesions such as point mutations. For example, EMS alkylates guanine bases, which results in base mispairing: an alkylated guanine will pair with a thymine base, resulting primarily in G/C to A/T transitions. Following mutagenesis, cereal plants are regenerated from the treated cells using known techniques. For instance, the resulting cereal seeds may be planted in accordance with conventional growing procedures and following self- pollination seed is formed on the plants. Additional seed that is formed as a result of such self- pollination in the present or a subsequent generation may be harvested and screened for the pres- ence of mutant Rca alleles. Several techniques are known to screen for specific mutant alleles, e.g., Deleteagene™ (Delete -a-gene; Li et al., 2001, Plant J 27: 235-242) uses polymerase chain reaction (PCR) assays to screen for deletion mutants generated by fast neutron mutagenesis, TILLING (tar- geted induced local lesions in genomes; McCallum et al., 2000, Nat Biotechnol 18:455-457) iden- tifies EMS-induced point mutations, etc.
[40] Gene editing, as used herein, refers to the targeted modification of genomic DNA using se- quence-specific enzymes (such as endonuclease, nickases, base conversion enzymes) and/or donor nucleic acids (e.g. dsDNA, oligo’s) to introduce desired changes in the DNA. Sequence-specific nucleases that can be programmed to recognize specific DNA sequences include meganucleases (MGNs), zinc-finger nucleases (ZFNs), TAL-effector nucleases (TALENs) and RNA-guided or DNA-guided nucleases such as Cas9, Cpfl, CasX, CasY, C2cl, C2c3, certain Argonaut-based sys- tems (see e.g. Osakabe and Osakabe, Plant Cell Physiol. 2015 Mar; 56(3):389-400; Ma et al., Mol Plant. 2016 Jul 6;9(7):961-74; Bortesie et al., Plant Biotech J, 2016, 14; Murovec et al., Plant Biotechnol J. 2017 Apr 1; Nakade et al., Bioengineered 8-3, 2017; Burstein et al., Nature 542, 37- 241; Komor et al., Nature 533, 420-424, 2016; all incorporated herein by reference). Donor nucleic acids can be used as a template for repair of the DNA break induced by a sequence specific nuclease but can also be used as such for gene targeting (without DNA break induction) to introduce a de- sired change into the genomic DNA. Sequence-specific nucleases may also be used without donor nucleic acid, thereby allowing insertion or deletion mutations via non-homologous end joining re- pair mechanism. Gene editing can be used to create mutant Rca alleles.
[41 ] Mutant nucleic acid molecules or mutant alleles may comprise one or more mutations or mod- ifications, such as: a. a“missense mutation”, which is a change in the nucleic acid sequence that results in the substitution of an amino acid for another amino acid;
b. a“nonsense mutation” or“STOP codon mutation”, which is a change in the nucleic acid sequence that results in the introduction of a premature STOP codon and thus the termina- tion of translation (resulting in a truncated protein); plant genes contain the translation stop codons“TGA” (UGA in RNA),“TAA” (UAA in RNA) and“TAG” (UAG in RNA); thus any nucleotide substitution, insertion, deletion which results in one of these codons to be in the mature mRNA being translated (in the reading frame) will terminate translation; c. an“insertion mutation” of one or more amino acids, due to one or more codons having been added in the coding sequence of the nucleic acid;
d. a“deletion mutation” of one or more amino acids, due to one or more codons having been deleted in the coding sequence of the nucleic acid;
e. a“frameshift mutation”, resulting in the nucleic acid sequence being translated in a differ- ent frame downstream of the mutation. A frameshift mutation can have various causes, such as the insertion, deletion or duplication of one or more nucleotides; f. a mutated splice site, resulting in altered splicing, which results in an altered mRNA pro- cessing and, consequently, in an altered encoded protein which contains either deletions, substitutions or insertions of various lengths, possibly combined with premature translation termination. Rca proteins and nucleic acids
[42] Rca proteins are AAA+ chaperones which can form hexameric protein complexes and interact with Rubisco. Functional Rca proteins comprise a central ATPase domain (the AAA+ module) and a C-terminal domain involved in Rubisco-Rca and Rca-Rca interactions33-35. The AAA+ module is located from amino acid at a position equivalent to position 57 to a position equivalent to position 345 on SEQ ID NO: 5, the ATPase core is located from amino acid at a position equivalent to position 182 to a position equivalent to position 282 on SEQ ID NO: 5 and the C-terminal domain is located from amino acid at a position equivalent to position 346 to a position equivalent to posi- tion 427 on SEQ ID NO: 5. More specifically, functional Rca proteins furthermore comprise an N- linker (IA) at the amino acid positions equivalent to position 123 and 124 on SEQ ID NO: 5, a Walker A motif (GxxxxGK) at the amino acid positions equivalent to positions 155 to 161 on SEQ ID NO: 5, a Walker B motif (LxxxD) at the amino acid positions equivalent to positions 215 to 219 on SEQ ID NO: 5, a Rubisco interaction loop (Shivhare et al 2017) at the amino acid positions equivalent to positions 253 to 265 on SEQ ID NO: 5, an Rca-Rca interface (Stotz et al. 2011, Nature Structural and Molecular Biology 18: 1366-1370) at the amino acid positions equivalent to posi- tions 339 to 347 on SEQ ID NO: 5, and a tyrosine (Y) at the amino acid positions equivalent to position 406 on SEQ ID NO: 5.
[43] Complexes comprising the Rubisco Activase and the Rubisco protein may be formed in vitro or in vivo. For example, the complexes may be formed in vitro by contacting a 382R Rca protein according to the invention with Rubisco from wheat, rice, Brassica, cotton, maize, soybean, Ara- bidopsis, or other plant species present in a leaf extract or Rubisco that has been purified from wheat, rice, Brassica, cotton, maize, soybean, Arabidopsis, or other plant species. Alternatively, the complexes may be formed in vivo by expression of an Rca polypeptide of the invention in a plant such that it forms a complex with the endogenous Rubisco of the plant.
[44] Any method known in the art can be used to assay the activity of complexes comprising Rca polypeptides (including, but not limited to, Rubisco activation and ATP hydrolysis, see for example Chakrabarti et al. 2002 J. Biochem. Biophys. Methods 52: 179-187 and McC Lilley and Portis 1997 Plant Physiol. 114:605-613).
Endogenous Rca gene
[45] As described herein, the endogenous Rca a protein may comprise the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 or 87. The endogenous Rca a protein may also comprise an amino acid sequence having at least 90% sequence identity with the amino acid se- quences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 or 87 and not comprise an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
[46] An endogenous Rca a protein may comprise an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% sequence identity to the above described protein which does not comprise an arginine at a position corresponding to position 382 of SEQ ID NO: 7. [47] SEQ ID NOs: 1 and 3 represent the amino acid sequences of the a isoform of the TaRca 2 A protein from wheat including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 5 and 7 represent the amino acid sequences of the TaRca 2a protein from the wheat sub- genome B including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 9 and 11 represent the amino acid sequences of the TaRca 2a protein from the wheat subgenome D including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 25 and 27 represent the amino acid sequences of the rice OsRca p arotein including and excluding, respec- tively, the chloroplast targeting peptide. SEQ ID NOs: 29 and 31 represent the amino acid se- quences of the BnRca 1a protein from the Brassica napus subgenome A including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 33 and 35 represent the amino acid sequences of the BnRca 2a protein from the Brassica napus subgenome A including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 37 and 39 represent the amino acid sequences of the BnRca 3a protein from the Brassica napus subgenome A including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 41 and 43 represent the amino acid sequences of the BnRca 1a protein from the Brassica napus subgenome C including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 45 and 47 represent the amino acid sequences of the BnRca 2a protein from the Brassica napus subgenome C including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 49 and 51 represent the amino acid sequences of the BnRca 3a protein from the Brassica napus subgenome C including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 53 and 55 represent the amino acid sequences of the GhRca 1a protein from the Gossypium hirsutum subgenome A including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 57 and 59 represent the amino acid sequences of the GhRca 2a protein from the Gossypium hirsutum subgenome A includ- ing and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 61 and 63 represent the amino acid sequences of the GhRca la protein from the Gossypium hirsutum subgenome D including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 65 and 67 represent the amino acid sequences of the GhRca 2a protein from the Gossypium hirsutum subge- nome D including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 69 and 71 represent the amino acid sequences of the soybean GmRca la protein including and exclud- ing, respectively, the chloroplast targeting peptide. SEQ ID NOs: 73 and 75 represent the amino acid sequences of the soybean GmRca 2a protein including and excluding, respectively, the chlo- roplast targeting peptide. SEQ ID NOs: 77 and 79 represent the amino acid sequences of the soy- bean GmRca 3a protein including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 81 and 83 represent the amino acid sequences of the maize ZmRca prote ain includ- ing and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 85 and 87 represent the amino acid sequences of the Arabidopsis AtRca pro atein including and excluding, respectively, the chloroplast targeting peptide. [48] Furthermore, the endogenous Rca genes encoding said endogenous Rca a proteins may com- prise the coding nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86 or 88.
[49] SEQ ID NOs: 2 and 4 represent the coding nucleotide sequences of the a isoform of the TaRca 2 A gene from wheat including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 6 and 8 represent the coding nucleotide sequences of the TaRca 2a gene from the wheat subgenome B including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 10 and 12 represent the coding nucleotide sequences of the TaRca 2a gene from the wheat subgenome D including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 26 and 28 represent the coding nucleotide sequences of the rice OsRca gene a including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 30 and 32 represent the coding nucleotide sequences of the BnRca 1 gen ae from the Brassica napus subgenome A including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 34 and 36 represent the coding nucleotide sequences of the BnRca 2a gene from the Brassica napus subgenome A in- cluding and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 38 and 40 represent the coding nucleotide sequences of the BnRca 3a gene from the Brassica napus subge- nome A including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 42 and 44 represent the coding nucleotide sequences of the BnRca la gene from the Brassica napus subgenome C including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 46 and 48 represent the coding nucleotide sequences of the BnRca 2a gene from the Brassica napus subgenome C including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 50 and 52 represent the coding nucleotide sequences of the BnRca 3a gene from the Brassica napus subgenome C including and excluding, respectively, the chloroplast targeting pep- tide. SEQ ID NOs: 54 and 56 represent the coding nucleotide sequences of the GhRca la gene from the Gossypium hirsutum subgenome A including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 58 and 60 represent the coding nucleotide sequences of the GhRca 2a gene from the Gossypium hirsutum subgenome A including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 62 and 64 represent the coding nucleotide sequences of the GhRca la gene from the Gossypium hirsutum subgenome D including and excluding, re- spectively, the chloroplast targeting peptide. SEQ ID NOs: 66 and 68 represent the coding nucleo- tide sequences of the GhRca 2a gene from the Gossypium hirsutum subgenome D including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 70 and 72 represent the coding nucleotide sequences of the soybean GmRca la gene including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 74 and 76 represent the coding nucleotide se- quences of the soybean GmRca 2a gene including and excluding, respectively, the chloroplast tar- geting peptide. SEQ ID NOs: 78 and 80 represent the coding nucleotide sequences of the soybean GmRca 3a gene including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 82 and 84 represent the coding nucleotide sequences of the maize ZmRca gen ae including and excluding, respectively, the chloroplast targeting peptide. SEQ ID NOs: 86 and 88 represent the coding nucleotide sequences of the Arabidopsis AtRca gen ae including and excluding, respec- tively, the chloroplast targeting peptide.
[50] The endogenous Rca a gene encoding said endogenous Rca a gene may also comprise a coding nucleotide sequence having at least 60% identity with the nucleotide sequence of SEQ ID NOs: 2,
4, 6, 8, 10, 12, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,
70, 72, 74, 76, 78, 80, 82, 84, 86 or 88 and not encoding an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
[51] A nucleic acid comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86 or 88 and not encoding an arginine at a position corresponding to position 382 of SEQ ID NO: 7 may be a nucleic acid comprising a nucleotide sequence having at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or 100% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,
82, 84, 86 or 88 and not encoding an arginine at a position corresponding to position 382 of SEQ ID NO: 7. Rca a knock-out alleles
[52] A knock-out allele of an Rca a gene is disclosed herein. The knock-out allele of an Rca a gene may be a full knock-out allele or a partial knock-out allele.
[53] A“full knock-out” or“null” allele, as used herein, refers to a mutant allele, which encodes a protein having no biological activity as compared to the corresponding wild-type functional protein or which encodes no protein at all. Such a“full knock-out mutant allele” is, for example, a wild- type allele, which comprises one or more mutations in its nucleic acid sequence, for example, one or more non-sense, mis-sense, insertion, deletion, frameshift or mutated splice site mutations. In particular, such a full knock-out mutant Rca a allele is a wild-type Rca a allele, which comprises a mutation that preferably result in the production of an Rca a protein lacking at least one functional domain or motif, such as the central ATPase domain (the AAA+ module), the C-terminal domain, the N-linker, Walker A, Walker B motives, the Rubisco interaction loop, the Rca-Rca interface, or lacking at least one amino acid critical for its function such as the tyrosine (Y) at the amino acid positions equivalent to position 406 of SEQ ID NO: 5, such that the biological activity of the Rca a protein is completely abolished, or whereby the modification(s) preferably result in no production of an Rca a protein.
[54] A“partial knock-out” mutant allele, as used herein, refers to a mutant allele, which encodes a protein having a significantly reduced biological activity as compared to the corresponding wild- type functional protein. Such a“partial knock-out mutant allele” is, for example, a wild-type allele, which comprises one or more mutations in its nucleic acid sequence, for example, one or more missense mutations. In particular, such a partial knockout mutant allele is a wild-type allele, which comprises a mutation that preferably results in the production of a protein wherein at least one conserved and/or functional amino acid is substituted for another amino acid, such that the biolog- ical activity is significantly reduced but not completely abolished.
[55] A missense mutation in an Rca a allele, as used herein, is any mutation (deletion, insertion or substitution) in an Rca a allele whereby one or more codons are changed into the coding DNA and the corresponding mRNA sequence of the corresponding wild type Rca a allele , resulting in the substitution of one or more amino acids in the wild type Rca a protein for one or more other amino acids in the mutant Rca a protein. A mutant Rca a allele comprising a missense mutation is an Rca a allele wherein one amino acid is substituted.
[56] A nonsense mutation in an Rca all aele, as used herein, is a mutation in an Rca allele w ahereby one or more translation stop codons are introduced into the coding DNA and the corresponding mRNA sequence of the corresponding wild type Rca a allele . Translation stop codons are TGA (UGA in the mRNA), TAA (UAA) and TAG (UAG). Thus, any mutation (deletion, insertion or substitution) that leads to the generation of an in-frame stop codon in the coding sequence will result in termination of translation and truncation of the amino acid chain. The truncated protein lacks the amino acids encoded by the coding DNA downstream of the mutation (i.e. the C-terminal part of the Rca a protein) and maintains the amino acids encoded by the coding DNA upstream of the mutation (i.e. the N-terminal part of the Rca a protein). The more truncated the mutant Rca a protein is in comparison to the wild type Rca a protein, the more the truncation may result in a significantly reduced activity of the Rca a protein. It is believed that, in order for the mutant Rca a protein to lose some biological activity, it should at least no longer comprise the tyrosine (Y) at the amino acid positions equivalent to position 406 of SEQ ID NO: 5.
Table 1 : Examples of substitution mutation resulting in the generation of an in-frame stop codon.
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
[57] A frameshift mutation in an Rca a allele , as used herein, is a mutation (deletion, insertion, duplication, and the like) in an Rca all aele that results in the nucleic acid sequence being translated in a different frame downstream of the mutation.
[58] A splice site mutation in an Rca a allele , as used herein, is a mutation (deletion, insertion, substitution, duplication, and the like) in an Rca a allele whereby a splice donor site or a splice acceptor site is mutated, resulting in altered processing of the mRNA and, consequently, an altered encoded protein, which can have insertions, deletions, substitutions of various lengths, or which can be truncated.
[59] A deletion mutation in an Rca a allele, as used herein, is a mutation in an Rca a allele that results in the production of an Rca a protein which lacks the amino acids encoded by the deleted coding DNA and maintains the amino acids encoded by the coding DNA upstream of the deletion (i.e. the N-terminal part of the Rca a protein) and encoding by the coding DNA downstream of the deletion (i.e. the C-terminal part of the Rca a protein).
[60] A“significantly reduced amount of functional Rca a protein” (e.g. functional wheat Rca a protein from the A, B or D subgenome, functional rice Rca a protein, functional Brassica Rca a protein from the A or C subgenome, functional cotton Rca a protein from the A or D subgenome, functional Soybean Rca a protein or functional com Rca a protein) refers to a reduction in the amount of a functional protein produced by the cell comprising a mutant Rca alle ale by at least 30%, 40%, 50%, 60%, 70%, or 80% as compared to the amount of the functional Rca a protein produced by the cell not comprising the mutant Rca a allele. The production of functional Rca a protein is however not abolished. This definition encompasses the production of a“non-functional” Rca a protein (e.g. truncated Rca a protein) having reduced biological activity in vivo, the reduction in the absolute amount of the functional Rca a protein (e.g. no functional Rca a protein being made due to the mutation in the Rca a gene), the production of an Rca a protein with significantly reduced biological activity compared to the activity of a functional wild type Rca a protein (such as an Rca a protein in which one or more amino acid residues that are crucial for the biological activity of the encoded Rca a protein are substituted for another amino acid residue).
Arginine Rca a variants
[61] In an embodiment, an“arginine Rca a protein variant” is provided. Such arginine Rca a protein variant is an Rca a protein wherein the amino acid at position corresponding to position 382 on SEQ ID NO: 7 is replaced with, or substituted with, an arginine.
[62] The arginine Rca a variant may comprise an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 3, 7, 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87, where the amino acid at position equivalent to position 382 of SEQ ID NO: 7 is substituted with an arginine or an amino acid sequence having at least 90% identity to the amino acid sequences of SEQ ID NOs: 3, 7, 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87 and comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
[63] The arginine Rca a protein variant may also comprise an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 3, 7, 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87, where the amino acid at position equivalent to position 382 of SEQ ID NO: 7 is substituted with an arginine, and further comprising a chloroplast targeting peptide; or an amino acid sequence having at least 90% identity to the amino acid sequences of SEQ ID NOs: 3, 7, 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87, further comprising a chloroplast targeting peptide and comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
[64] The arginine Rca a variant may comprise an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1, 5, 9, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 or 85, where the amino acid at position equivalent to position 382 of SEQ ID NO: 7 is substituted with an arginine; or an amino acid sequence having at least 90% identity to the amino acid se- quences of SEQ ID NOs: 1, 5, 9, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 or 85 and comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
[65] The arginine Rca a protein variant may comprise an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% sequence identity to the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 or 87 and comprise an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
[66] In addition, it is clear that arginine variants of Rca a proteins, wherein one or more amino acid residues have been deleted, substituted or inserted, can also be used to the same effect in the meth- ods according to the invention, provided that the central ATPase domain (the AAA+ module), the C-terminal domain, the N-linker, Walker A, Walker B motives, the Rubisco interaction loop, the Rca-Rca interface and the tyrosine (Y) at the amino acid positions equivalent to position 406 of SEQ ID NO: 5 are not affected by the deletion, substitution or insertion of amino-acid.
[67] Suitable for the invention are chloroplast targeting peptides which enable the subcellular tar- geting of the Rca proteins according to the invention to the chloroplast. Chloroplast transit peptide, chloroplast targeting sequence and stromal -targeting transit peptide are equivalent terms. Chloro- plast targeting peptides are recognizable based on the presence of three domains: an uncharged N- terminal domain of about 10 residues beginning with a methionine followed by an alanine and terminating with a glycine or a proline, a central domain lacking acidic residues but enriched in serines and threonines and a C-terminal domain enriched in arginines and forming an amphiphilic b strand (Bruce, 2000, trends in cell biology, Vol 10, 440-447).
[68] Such chloroplast targeting peptides are identified herein as the amino acid sequence from po- sition 1 to position 46 of SEQ ID NOs: 1, 9 or 25, the amino acid sequence from position 1 to position 48 of SEQ ID NO: 5, the amino acid sequence from position 1 to position 47 of SEQ ID NOs: 13, 17 or 21, the amino acid sequence from position 1 to position 59 of SEQ ID NO: 29 or 41, the amino acid sequence from position 1 to position 58 of SEQ ID NO: 33 or 45, the amino acid sequence from position 1 to position 52 of SEQ ID NO: 37 or 49, the amino acid sequence from position 1 to position 55 of SEQ ID NO: 53 or 61, the amino acid sequence from position 1 to position 51 of SEQ ID NO: 57 or 65, the amino acid sequence from position 1 to position 60 of SEQ ID NO: 69 or 77, the amino acid sequence from position 1 to position 57 of SEQ ID NO: 73, the amino acid sequence from position 1 to position 35 of SEQ ID NO: 81, the amino acid sequence from position 1 to position 58 of SEQ ID NO: 85.
[69] Also suitable for the invention are chloroplast targeting peptides having an amino acid se- quence having at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% identity to the amino acid sequence from position 1 to position 46 of SEQ ID NOs: 1, 9 or 25, the amino acid sequence from position 1 to position 48 of SEQ ID NO: 5, the amino acid sequence from position 1 to position 47 of SEQ ID NOs: 13, 17 or 21, the amino acid sequence from position 1 to position 59 of SEQ ID NO: 29 or 41, the amino acid sequence from position 1 to position 58 of SEQ ID NO: 33 or 45, the amino acid sequence from position 1 to position 52 of SEQ ID NO: 37 or 49, the amino acid sequence from position 1 to position 55 of SEQ ID NO: 53 or 61, the amino acid sequence from position 1 to position 51 of SEQ ID NO: 57 or 65, the amino acid sequence from position 1 to position 60 of SEQ ID NO: 69 or 77, the amino acid sequence from position 1 to position 57 of SEQ ID NO: 73, the amino acid sequence from position 1 to position 35 of SEQ ID NO: 81, the amino acid sequence from position 1 to position 58 of SEQ ID NO: 85. A chloroplast targeting peptide having an amino acid sequence having at least 80% sequence identity to the amino acid sequence from position 1 to position 46 of SEQ ID NOs: 1, 9 or 25, the amino acid sequence from position 1 to position 48 of SEQ ID NO: 5, the amino acid sequence from position 1 to position 47 of SEQ ID NOs: 13, 17 or 21, the amino acid sequence from position 1 to position 59 of SEQ ID NO: 29 or 41, the amino acid sequence from position 1 to position 58 of SEQ ID NO: 33 or 45, the amino acid sequence from position 1 to position 52 of SEQ ID NO: 37 or 49, the amino acid sequence from position 1 to position 55 of SEQ ID NO: 53 or 61, the amino acid sequence from position 1 to position 51 of SEQ ID NO: 57 or 65, the amino acid sequence from position 1 to position 60 of SEQ ID NO: 69 or 77, the amino acid sequence from position 1 to position 57 of SEQ ID NO: 73, the amino acid sequence from position 1 to position 35 of SEQ ID NO: 81, the amino acid sequence from position 1 to position 58 of SEQ ID NO: 85 can thus be a chloroplast targeting peptide having an amino acid sequence having at least at least 80%, or at least 85%, or at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or even 100% sequence identity to the amino acid sequence from position 1 to position 46 of SEQ ID NOs: 1, 9 or 25, the amino acid sequence from position 1 to position 48 of SEQ ID NO: 5, the amino acid sequence from position 1 to position 47 of SEQ ID NOs: 13, 17 or 21, the amino acid sequence from position 1 to position 59 of SEQ ID NO: 29 or 41, the amino acid sequence from position 1 to position 58 of SEQ ID NO: 33 or 45, the amino acid sequence from position 1 to position 52 of SEQ ID NO: 37 or 49, the amino acid sequence from position 1 to position 55 of SEQ ID NO: 53 or 61, the amino acid sequence from position 1 to position 51 of SEQ ID NO: 57 or 65, the amino acid sequence from position 1 to position 60 of SEQ ID NO: 69 or 77, the amino acid sequence from position 1 to position 57 of SEQ ID NO: 73, the amino acid sequence from position 1 to position 35 of SEQ ID NO: 81, the amino acid sequence from position 1 to position 58 of SEQ ID NO: 85.
[70] In another embodiment, a nucleic acid encoding an arginine Rca a protein variant is provided.
The nucleic acid encoding an arginine Rca a protein variant may comprise.
[71] A coding nucleotide sequence selected from (a) the coding nucleotide sequences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88, where the nucleotides encoding the amino acid at position equivalent to position 382 of SEQ ID NO: 7 are substituted with nucleotides encoding an arginine, or the complement thereof; (b) a coding nucleotide sequence having at least 60% identity to the coding nucleotide sequences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88 and encoding an arginine at position equivalent to position 382 of SEQ ID NO: 7; (c) the coding nucleotide sequences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88, where the nucleotides encoding the amino acid at position equivalent to position 382 of SEQ ID NO: 7 are substituted with nucleotides encoding an arginine, or the complement thereof, and further comprising a nucleic acid encoding a chloroplast targeting peptide; (d) a coding nucleotide sequence having at least 60% identity to the coding nucleotide sequences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88 and encoding an arginine at position equivalent to position 382 of SEQ ID NO: 7 and further comprising a nucleic acid encoding a chloroplast targeting peptide; (e) the coding nucleotide sequences of SEQ ID NOs: 2, 6, 10, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82 or 86, where the nucleotides encoding the amino acid at position equivalent to position 382 of SEQ ID NO: 7 are substituted with nucleotides encoding an arginine, or the complement thereof; (f) a coding nucleotide sequence having at least 60% identity to the coding nucleotide sequences of SEQ ID NOs: 2, 6, 10, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82 or 86 and encoding an arginine at position equivalent to position 382 of SEQ ID NO: 7.
[72] Suitable for the invention are nucleic acids encoding an arginine Rca a protein variant, which comprise a nucleotide sequence having at least 40%, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to the herein described gene and are also referred to as variants.
[73] A nucleic acid comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88 and encoding an arginine at position equivalent to position 382 of SEQ ID NO: 7 can thus be a nucleic acid com- prising a nucleotide sequence having at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or 100% sequence identity to SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88 and encoding an arginine at position equivalent to position 382 of SEQ ID NO: 7.
[74] Also suitable for the invention are nucleic acid encoding a chloroplast targeting peptide having the coding nucleotide sequence from position 1 to position 138 of SEQ ID NOs: 2, 10 or 26, the coding nucleotide sequence from position 1 to position 144 of SEQ ID NO: 6, the coding nucleotide sequence from position 1 to position 141 of SEQ ID NOs: 14, 18 or 22, the coding nucleotide sequence from position 1 to position 177 of SEQ ID NO: 30 or 42, the coding nucleotide sequence from position 1 to position 174 of SEQ ID NO: 34 or 46, the coding nucleotide sequence from position 1 to position 156 of SEQ ID NO: 38 or 50, the coding nucleotide sequence from position 1 to position 165 of SEQ ID NO: 54 or 62, the coding nucleotide sequence from position 1 to position 153 of SEQ ID NO: 58 or 66, the coding nucleotide sequence from position 1 to position 180 of SEQ ID NO: 70 or 78, the coding nucleotide sequence from position 1 to position 171 of SEQ ID NO: 74, the coding nucleotide sequence from position 1 to position 105 of SEQ ID NO: 82, the coding nucleotide sequence from position 1 to position 174 of SEQ ID NO: 86.
[75] Also suitable to the invention are nucleic acid encoding a chloroplast targeting peptide having a coding nucleotide sequence having at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or 100% sequence identity to the coding nucleotide sequence from position 1 to position 138 of SEQ ID NOs: 2, 10 or 26, the coding nucleotide se- quence from position 1 to position 144 of SEQ ID NO: 6, the coding nucleotide sequence from position 1 to position 141 of SEQ ID NOs: 14, 18 or 22, the coding nucleotide sequence from position 1 to position 177 of SEQ ID NO: 30 or 42, the coding nucleotide sequence from position 1 to position 174 of SEQ ID NO: 34 or 46, the coding nucleotide sequence from position 1 to position 156 of SEQ ID NO: 38 or 50, the coding nucleotide sequence from position 1 to position 165 of SEQ ID NO: 54 or 62, the coding nucleotide sequence from position 1 to position 153 of SEQ ID NO: 58 or 66, the coding nucleotide sequence from position 1 to position 180 of SEQ ID NO: 70 or 78, the coding nucleotide sequence from position 1 to position 171 of SEQ ID NO: 74, the coding nucleotide sequence from position 1 to position 105 of SEQ ID NO: 82, the coding nucleotide sequence from position 1 to position 174 of SEQ ID NO: 86.
Arginine Rca a allele
[76] The Arginine Rca a protein variant may be encoded by an“arginine allele” of an Rca a gene.
[77] In a further embodiment, said arginine allele may comprise (a) the coding nucleotide se- quences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88, where the nucleotides encoding the amino acid at position equivalent to position 382 of SEQ ID NO: 7 are substituted with nucleotides encoding an arginine, or the complement thereof; (b) a cod- ing nucleotide sequence having at least 60% identity to the coding nucleotide sequences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88 and encoding an arginine at position equivalent to position 382 of SEQ ID NO: 7; (c) the coding nucleotide se- quences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88, where the nucleotides encoding the amino acid at position equivalent to position 382 of SEQ ID NO: 7 are substituted with nucleotides encoding an arginine, or the complement thereof, and further comprising a nucleic acid encoding a chloroplast targeting peptide; (d) a coding nucleotide se- quence having at least 60% identity to the coding nucleotide sequences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88 and encoding an arginine at position equivalent to position 382 of SEQ ID NO: 7 and further comprising a nucleic acid encoding a chloroplast targeting peptide; (e) the coding nucleotide sequences of SEQ ID NOs: 2, 6, 10, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82 or 86, where the nucleotides encoding the amino acid at position equivalent to position 382 of SEQ ID NO: 7 are substituted with nucleotides en- coding an arginine, or the complement thereof; (f) a coding nucleotide sequence having at least 60% identity to the coding nucleotide sequences of SEQ ID NOs: 2, 6, 10, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82 or 86 and encoding an arginine at position equivalent to position 382 of SEQ ID NO: 7.
[78] Using the technologies of gene editing, endogenous alleles in a plant encoding a Rca a protein can be converted to an arginine Rca a allele by making the desired changes (missense mutations) to existing Rca g aenes, or by replacing one or more endogenous sequences encoding Rca a pro- teins with sequences encoding arginine Rca a protein variants, e.g. as described herein (deletion and insertion mutations).
[79] An endogenous allele in a plant, such as a cereal plant, wheat, com, Brassica, rice, cotton or soybean, encoding a Rca a protein can also be converted to an arginine Rca a allele by making the desired changes (missense mutations) to existing Rca ge anes using mutagenesis.
Recombinant genes and vectors
[80] In yet another aspect, a recombinant gene is provided comprising the following operably linked elements (a) a promoter, preferably expressible in plants, (b) a nucleic acid encoding a Ar- ginine Rca a protein and, optionally (c) a transcription termination and polyadenylation region, preferably a transcription termination and polyadenylation region functional in plants. In a further embodiment, the Arginine Rca a protein and nucleic acid variants comprise an amino acid se- quences and nucleotide sequences according to the invention. In a further embodiment, said pro- moter is a constitutive promoter, tissue-specific promoter or an inducible promoter. In yet a further embodiment, the promoter may be a green tissue specific promoter, a mesophyll specific promoter, a light-induced promoter or a temperature induced promoter.
[81] Furthermore, a recombinant gene capable of suppressing specifically the expression of the endogenous Rca a genes is described which comprises the following operably linked elements (a) a promoter, preferably expressible in plants, (b) a nucleic acid which when transcribed yields an RNA molecule inhibitory to the endogenous Rca a genes encoding a Rca a protein not comprising an arginine at a position corresponding to position 382 of SEQ ID NO: 7 but not inhibitory to genes encoding Arginine Rca a protein variants; and, optionally (c) a transcription termination and poly- adenylation region, preferably a transcription termination and polyadenylation region functional in plants. The endogenous Rca a genes may comprise the coding nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86 or 88 or a coding nucleotide sequence having at least 60% identity with the nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86 or 88 and not encoding an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
[82] Such inhibitory RNA molecule can reduce the expression of a gene for example through the mechanism of RNA-mediated gene silencing. It can be a silencing RNA downregulating expression of a target gene. As used herein,“silencing RNA” or“silencing RNA molecule” refers to any RNA molecule, which upon introduction into a plant cell, reduces the expression of a target gene. Such silencing RNA may e.g. be so-called“antisense RNA”, whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the complement of the sequence of the target nucleic acid, preferably the coding sequence of the target gene. How- ever, antisense RNA may also be directed to regulatory sequences of target genes, including the promoter sequences and transcription termination and polyadenylation signals. Silencing RNA fur- ther includes so-called“sense RNA” whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the sequence of the target nucleic acid. Other silencing RNA may be“unpolyadenylated RNA” comprising at least 20 consecutive nucle- otides having 95% sequence identity to the complement of the sequence of the target nucleic acid, such as described in WOOl/12824 or US6423885 (both documents herein incorporated by refer- ence). Yet another type of silencing RNA is an RNA molecule as described in W003/076619 (herein incorporated by reference) comprising at least 20 consecutive nucleotides having 95% se- quence identity to the sequence of the target nucleic acid or the complement thereof, and further comprising a largely-double stranded region as described in W003/076619 (including largely dou- ble stranded regions comprising a nuclear localization signal from a viroid of the Potato spindle tuber viroid-type or comprising CUG trinucleotide repeats). Silencing RNA may also be double stranded RNA comprising a sense and antisense strand as herein defined, wherein the sense and antisense strand are capable of base-pairing with each other to form a double stranded RNA region (preferably the said at least 20 consecutive nucleotides of the sense and antisense RNA are com- plementary to each other). The sense and antisense region may also be present within one RNA molecule such that a hairpin RNA (hpRNA) can be formed when the sense and antisense region form a double stranded RNA region. hpRNA is well-known within the art (see e.g W099/53050, herein incorporated by reference). The hpRNA may be classified as long hpRNA, having long, sense and antisense regions which can be largely complementary, but need not be entirely comple- mentary (typically larger than about 200 bp, ranging between 200-1000 bp). hpRNA can also be rather small ranging in size from about 30 to about 42 bp, but not much longer than 94 bp (see W004/073390, herein incorporated by reference). Silencing RNA may also be artificial micro- RNA molecules as described e.g. in W02005/052170, W02005/047505 or US 2005/0144667, or ta-siRNAs as described in W02006/074400 (all documents incorporated herein by reference). Said RNA capable of modulating the expression of a gene can also be an RNA ribozyme. [83] The phrase "operably linked" refers to the functional spatial arrangement of two or more nu- cleic acid regions or nucleic acid sequences. For example, a promoter region may be positioned relative to a nucleic acid sequence such that transcription of a nucleic acid sequence is directed by the promoter region. Thus, a promoter region is "operably linked" to the nucleic acid sequence. “Functionally linked” is an equivalent term.
[84] A“transcription termination and polyadenylation region” as used herein is a sequence that controls the cleavage of the nascent RNA, whereafter a poly(A) tail is added at the resulting RNA 3’ end, functional in plant cells. Transcription termination and polyadenylation signals functional in plant cells include, but are not limited to, 3’nos, 3’35S, 3’his and 3’g7.
[85] As used herein, the term "plant-expressible promoter" means a DNA sequence that is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin such as the CaMV35S (Harpster et l. (198 a8) Mol Gen Genet. 212(1): 182-90, the subterranean clover virus promoter No 4 or No 7 (WO9606932), or T-DNA gene promoters but also tissue-specific or organ-specific promoters including but not lim- ited to seed-specific promoters (e.g., WO89/03887), organ-primordia specific promoters (An et l. (1996) Plant Cell 8(1): 15-30), stem-specific promoters (Keller et l., (19 a88 ) EMBO J. 7(12): 3625- 3633), leaf specific promoters (Hudspeth et l. a (1989) Plant Mol Biol. 12: 579-589), mesophyl- specific promoters (such as the light-inducible Rubisco promoters), root-specific promoters (Keller et al. (1989) Genes Dev. 3: 1639-1646), tuber-specific promoters (Keil et l. (198 a9) EMBO J. 8(5): 1323-1330), vascular tissue specific promoters (Peleman et al. (1989) Gene 84: 359-369), stamen- selective promoters (WO 89/10396, WO 92/13956), dehiscence zone specific promoters (WO 97/13865) and the like.
[86] Suitable promoters for the invention are constitutive plant-expressible promoters. Constitutive plant-expressible promoters are well known in the art and include the CaMV35S promoter (Harp- ster et l a. (1988) Mol Gen Genet. 212(1): 182-90), Actin promoters, such as, for example, the pro- moter from the Rice Actin gene (McElroy et al., 1990, Plant Cell 2: 163), the promoter of the Cas- sava Vein Mosaic Virus (Verdaguer et al., 1996 Plant Mol. Biol. 31: 1129), the GOS promoter (de Pater et al., 1992, Plant J. 2:837), the Histone H3 promoter (Chaubet et al., 1986, Plant Mol Biol 6:253), the Agrobacterium tumefaciens Nopaline Synthase (Nos) promoter (Depicker et al., 1982, J. Mol. Appl. Genet. 1: 561), or Ubiquitin promoters, such as, for example, the promoter of the maize Ubiquitin-1 gene (Christensen et al., 1992, Plant Mol. Biol. 18:675).
[87] A further promoter suitable for the invention is the endogenous promoter driving expression of the gene encoding an Rca protein. [88] Any of the nucleic acid sequences described above may be provided in a recombinant vector. A recombinant vector typically comprises, in a 5' to 3' orientation: a promoter to direct the tran- scription of a nucleic acid sequence and a nucleic acid sequence. The recombinant vector may further comprise a 3' transcriptional terminator, a 3' polyadenylation signal, other untranslated nu- cleic acid sequences, transit and targeting nucleic acid sequences, selectable markers, enhancers, and operators, as desired. The wording "5' UTR" refers to the untranslated region of DNA upstream, or 5' of the coding region of a gene and "3' UTR" refers to the untranslated region of DNA down- stream, or 3' of the coding region of a gene. Means for preparing recombinant vectors are well known in the art. Methods for making recombinant vectors particularly suited to plant transfor- mation are described in US4971908, US4940835, US4769061 and US4757011. Typical vectors useful for expression of nucleic acids in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens . One or more addi- tional promoters may also be provided in the recombinant vector. These promoters may be operably linked, for example, without limitation, to any of the nucleic acid sequences described above. Al- ternatively, the promoters may be operably linked to other nucleic acid sequences, such as those encoding transit peptides, selectable marker proteins, or antisense sequences. These additional pro- moters may be selected on the basis of the cell type into which the vector will be inserted. Also, promoters which function in bacteria, yeast, and plants are all well taught in the art. The additional promoters may also be selected on the basis of their regulatory features. Examples of such features include enhancement of transcriptional activity, inducibility, tissue specificity, and developmental stage-specificity.
[89] The recombinant vector may also contain one or more additional nucleic acid sequences. These additional nucleic acid sequences may generally be any sequences suitable for use in a recombinant vector. Such nucleic acid sequences include, without limitation, any of the nucleic acid sequences, and modified forms thereof, described above. The additional structural nucleic acid sequences may also be operably linked to any of the above described promoters. The one or more structural nucleic acid sequences may each be operably linked to separate promoters. Alternatively, the structural nucleic acid sequences may be operably linked to a single promoter (i.e. a single operon).
Methods and uses
[90] In one aspect, the invention provides a method for increasing the ratio of an arginine Rca a protein variant in plants comprising (a) providing to cells of a plant the recombinant gene of the invention; and reducing the expression of endogenous Rca a protein in said plant cells, wherein said ratio is increased compared to a control plant cell not comprising said recombinant gene; or (b) introducing into cells of a plant at least one arginine Rca a allele according to the invention, wherein said ratio is increased compared to a control plant cell not comprising said arginine Rca a allele allele. In a further embodiment, the cereal plant is a wheat plant, a Brassica plant, a cotton plant, a maize plant, a soybean plant, a rice plant or an Arabidopsis plant.
[91] In another embodiment, said Rca a protein variants comprise an amino acid sequence as de- scribed above and is encoded by nucleic acids comprising the coding nucleic acid sequences de- scribed above.
[92] “Increasing the ratio of an arginine Rca a protein variant” as used herein mean increasing the relative abundance of arginine Rca a protein variants over the overall abundance of Rca a proteins. This can be achieved by increasing the abundance of arginine Rca a protein variants, by decreasing the abundance of endogenous Rca a proteins and/or by both increasing the abundance of arginine Rca a protein variants and decreasing the abundance of endogenous Rca a proteins. The increased ratio of arginine Rca a protein variants may be of at least about or about 15%, at least about or about 30%, at least about or about 45%, at least about or about 60%, at least about or about 75%, at least about or about 90%, or at least about or about 100%. The increased ratio of arginine Rca a protein variants may be between about 15% and about 30%, between about 15% and about 45%, between about 15% and about 60%, between about 15% and about 75%, between about 15% and about 90%, between about 15% and about 100%, between about 30% and about 45%, between about 30% and about 60%, between about 30% and about 75%, between about 30% and about 90%, between about 30% and about 100%, between about 45% and about 60%, between about 45% and about 75%, between about 45% and about 90%, between about 45% and about 100%, between about 60% and about 75%, between about 60% and about 90%, between about 60% and about 100%, between about 75% and about 90%, between about 75% and about 100%, between about 90% and about 100%.
[93] “Introducing” in connection with the present application relates to the placing of genetic in- formation in a plant cell or plant by artificial means. This can be effected by any method known in the art for introducing RNA or DNA into plant cells, protoplasts, calli, roots, tubers, seeds, stems, leaves, seedlings, embryos, pollen and microspores, other plant tissues, or whole plants. "Introduc- ing" also comprises stably integrating into the plant's genome. Introducing the recombinant gene can be performed by transformation or by crossing with a plant obtained by transformation or its descendant (also referred to as“introgression”). Introducing an allele also may be performed by mutagenesis of by gene editing.
[94] The term“providing” may refer to introduction of an exogenous DNA molecule to a plant cell by transformation, optionally followed by regeneration of a plant from the transformed plant cell. The term may also refer to introduction of the recombinant DNA molecule by crossing of a trans- genic plant comprising the recombinant DNA molecule with another plant and selecting progeny plants which have inherited the recombinant DNA molecule or transgene. Yet another alternative meaning of providing refers to introduction of the recombinant DNA molecule by techniques such as protoplast fusion, optionally followed by regeneration of a plant from the fused protoplasts.
[95] The recombinant gene may be provided to a plant cell by methods well-known in the art.
[96] The term "transformation" herein refers to the introduction (or transfer) of nucleic acid into a recipient host such as a plant or any plant parts or tissues including plant cells, protoplasts, calli, roots, tubers, seeds, stems, leaves, fibers, seedlings, embryos and pollen. Plants containing the transformed nucleic acid sequence are referred to as“transgenic plants”. Transformed, transgenic and recombinant refer to a host organism such as a plant into which a heterologous nucleic acid molecule (e.g. an expression cassette or a recombinant vector) has been introduced. The nucleic acid can be stably integrated into the genome of the plant.
[97] As used herein, the phrase "transgenic plant" refers to a plant having a nucleic acid stably integrated into a genome of the plant, for example, the nuclear or plastid genomes. In other words, plants containing transformed nucleic acid sequence are referred to as "transgenic plants" and in- cludes plants directly obtained from transformation and their descendants (Tx generations). Trans- genic and recombinant refer to a host organism such as a plant into which a heterologous nucleic acid molecule (e.g. the promoter, the recombinant gene or the vector as described herein) has been introduced. The nucleic acid can be stably integrated into the genome of the plant.
[98] It will be clear that the methods of transformation used are of minor relevance to the current invention. Transformation of plants is now a routine technique. Advantageously, any of several transformation methods may be used to introduce the nucleic acid/gene of interest into a suitable ancestor cell. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens et al. (1982) Nature 296: 72-74 ; Negru- tiu et al. (1987) Plant. Mol. Biol. 8: 363-373); electroporation of protoplasts (Shillito et al. (1985) Bio/Technol. 3: 1099-1102); microinjection into plant material (Crossway et al. (1986) Mol. Gen. Genet. 202: 179-185); DNA or RNA-coated particle bombardment (Klein et al. (1987) Nature 327: 70) infection with (non-integrative) viruses and the like.
[99] Different transformation systems could be established for various cereals: the electroporation of tissue, the transformation of protoplasts and the DNA transfer by particle bombardment in re- generable tissue and cells (for an overview see Jane, Euphytica 85 (1995), 35-44). The transfor- mation of wheat has been described several times in literature (for an overview see Maheshwari, Critical Reviews in Plant Science 14 (2) (1995), 149-178, Nehra et al., Plant J. 5 (1994), 285-297). [ 100] The recombinant DNA molecules according to the invention may be provided to plants in a stable manner or in a transient manner using methods well known in the art. The recombinant genes may be introduced into plants or may be generated inside the plant cell as described e.g. in EP 1339859.
[101] “Control plant” as used herein refers to a plant genetically resembling the tested plant but not carrying the recombinant gene, such as wild type plants or null segregant plants, or not carrying the mutant allele, such as wild type plants or wild type segregant plants.
[ 102] The transformed plant cells and plants obtained by the methods described herein may be fur- ther used in breeding procedures well known in the art, such as crossing, selfing, and backcrossing. Breeding programs may involve crossing to generate an FI (first filial) generation, followed by several generations of selfing (generating F2, F3, etc). The breeding program may also involve backcrossing (BC) steps, whereby the offspring is backcrossed to one of the parental lines, termed the recurrent parent.
[103] The transformed plant cells and plants obtained by the methods disclosed herein may also be further used in subsequent transformation procedures, e. g. to introduce a further recombinant gene.
[ 104] In a further embodiment, reducing the expression of endogenous Rca proteins comprises in- troducing into cells of the plant at least one knock-out mutant Rca allele according to the invention, or providing said cells of a plant with a second recombinant gene capable of suppressing specifi- cally the expression of the endogenous Rca a gene, as described above.
[105] Suitable for the invention are methods for increasing the ratio of an arginine Rca a protein variants in plants comprising introducing into cells of a wheat plant at least two, at least three, at least four, at least five or even all six knock-out mutant Rca a allele s according to the invention. Such at least two knock-out mutant Rca a allele s in wheat may be two knock-out mutant Rca a alleles from the subgenome B, two knock-out mutant Rca a allele s from the subgenome D, two knock-out mutant Rca a allele s from the subgenome A, one knock-out mutant Rca a allele from the subgenome B and one knock-out mutant Rca a allele from the subgenome D, one knock-out mutant Rca a allele from the subgenome B and one knock-out mutant Rca a allele from the subge- nome A or one knock-out mutant Rca a allele from the subgenome D and one knock-out mutant Rca a allele from the subgenome A. Such at least three knock-out mutant Rca a allele s in wheat may be two knock-out mutant Rca a alleles from the subgenome B and one knock-out mutant Rca a allele from the subgenome A, two knock-out mutant Rca a allele s from the subgenome B and one knock-out mutant Rca a allele from the subgenome D, two knock-out mutant Rca a allele s from the subgenome D and one knock-out mutant Rca a allele from the subgenome B, two knock- out mutant Rca a allele s from the subgenome D and one knock-out mutant Rca a allele from the subgenome A, two knock-out mutant Rca a alleles from the subgenome A and one knock-out mu- tant Rca a allele from the subgenome B, two knock-out mutant Rca a alleles from the subgenome A and one knock-out mutant Rca a allele from the subgenome D or one knock-out mutant Rca a alleles from the subgenome B, one knock-out mutant Rca a allele from the subgenome A and one knock-out mutant Rca a allele from the subgenome D.
[ 106] Such at least four knock-out mutant Rca a alleles in wheat may be two knock-out mutant Rca a alleles from the subgenome B and two knock-out mutant Rca a allele s from the subgenome A, two knock-out mutant Rca a alleles from the subgenome B and two knock-out mutant Rca a allele from the subgenome D, or two knock-out mutant Rca a allele s from the subgenome D and two knock-out mutant Rca a allele from the subgenome A. Such at least four knock-out mutant Rca a alleles may also be two knock-out mutant Rca a allele s from the subgenome B, one knock-out mutant Rca a allele s from the subgenome A and one knock-out mutant Rca a allele s from the subgenome D, or two knock-out mutant Rca alle ales from the subgenome D, one knock-out mutant Rca a alleles from the subgenome A and one knock-out mutant Rca a alleles from the subgenome B, or two knock-out mutant Rca a allele s from the subgenome A, one knock-out mutant Rca a alleles from the subgenome B and one knock-out mutant Rca allel aes from the subgenome D. Such at least five knock-out mutant Rca a allele s may be two knock-out mutant Rca a allele s from the subgenome B, two knock-out mutant Rca a alleles from the subgenome A and one knock-out mu- tant Rca a allele from the subgenome D, or two knock-out mutant Rca allele as from the subgenome B, two knock-out mutant Rca a allele s from the subgenome D and one knock-out mutant Rca a allele from the subgenome A, or two knock-out mutant Rca a allele s from the subgenome D, two knock-out mutant Rca a alleles from the subgenome A and one knock-out mutant Rca allele a from the subgenome B.
[ 107] Suitable for the invention are methods for increasing the ratio of an arginine Rca a protein variants in plants comprising introducing into cells of a Brassica plant at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or even all twelve knock-out mutant Rca a alleles according to the invention.
[108] Such at least two knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 alleles from the subgenome A, two knock-out mutant Rca 2 alleles from the subgenome A, two knock-out mutant Rca 3 alleles from the subgenome A, two knock-out mutant Rca 1 alleles from the subgenome C, two knock-out mutant Rca 2 alleles from the subgenome C, two knock-out mu- tant Rca 3 alleles from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, one knock- out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subge- nome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C.
[ 109] Such at least three knock-out mutant in Brassica may be two knock-out mutant Rca 1 alleles from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A, two knock- out mutant Rca 1 alleles from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 alleles from the subgenome A and one knock-out mu- tant Rca 1 allele from the subgenome C, two knock-out mutant Rca 1 alleles from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 1 alleles from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 2 alleles from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 alleles from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 alleles from the subge- nome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca
2 alleles from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 2 alleles from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 3 alleles from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 alleles from the subge- nome A and one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca
3 alleles from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 alleles from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 alleles from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 1 alleles from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 alleles from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 alleles from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 alleles from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 1 alleles from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 2 alleles from the subgenome C and one knock-out mutant Rca 1 allele from the sub- genome A, two knock-out mutant Rca 2 alleles from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 alleles from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 alleles from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C, two knock- out mutant Rca 2 alleles from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 3 alleles from the subgenome C and one knock-out mu- tant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 alleles from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 alleles from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 alleles from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 alleles from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C.
[1 10] Such at least three knock-out mutant in Brassica may also be one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A , one knock-out mutant Rca 1 allele from the subgenome A , one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock- out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subge- nome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock- out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subge- nome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca
2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock- out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subge- nome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca
3 allele from the subgenome C.
[ 111] Such at least four knock-out mutant Rca alleles in Brassica may be one knock-out mutant Rca
1 allele from the subgenome A , one knock-out mutant Rca 2 allele from the subgenome A ,one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A , one knock-out mutant Rca 2 allele from the subgenome A , one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A , one knock-out mutant Rca 2 allele from the subgenome A ,one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the sub- genome C; one knock-out mutant Rca 1 allele from the subgenome A , one knock-out mutant Rca
2 allele from the subgenome A one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A , one knock-out mutant Rca 2 allele from the subgenome A , one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A , one knock-out mutant Rca 2 allele from the subgenome A , one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A , one knock-out mutant Rca 3 allele from the subgenome A one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; one knock- out mutant Rca 1 allele from the subgenome A , one knock-out mutant Rca 3 allele from the sub- genome A , one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A , one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 2 allele from the subgenome A , one knock-out mutant Rca 3 allele from the subgenome A , one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; one knock-out mutant Rca 2 allele from the subgenome A , one knock-out mutant Rca 3 allele from the subgenome A , one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 2 allele from the subgenome A , one knock-out mutant Rca 3 allele from the subgenome A , one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 2 allele from the subgenome A , one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 3 allele from the subgenome A , one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subge- nome C.
[1 12] Such at least four knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock- out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subge- nome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C.
[1 13] Such at least four knock-out mutant Rca alleles in Brassica may furthermore be two knock- out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subge- nome C, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome A and two knock- out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subge- nome A and two knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C.
[1 14] Such at least five knock-out mutant Rca alleles in Brassica may be one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C.
[1 15] Such at least five knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subge- nome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subge- nome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock- out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the sub- genome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock- out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subge- nome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock- out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subge- nome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A.
[1 16] Such at least five knock-out mutant Rca alleles in Brassica may furthermore be two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome A and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subge- nome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock- out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the sub- genome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subge- nome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock- out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 1 allele from the sub- genome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subge- nome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subge- nome A; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C.
[1 17] Such at least six knock-out mutant Rca alleles in Brassica may be one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C,one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C.
[1 18] Such at least six knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A.
[1 19] Such at least six knock-out mutant Rca alleles in Brassica may furthermore be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subge- nome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the sub- genome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A;two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the sub- genome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock- out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the sub- genome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subge- nome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca
2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock- out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subge- nome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca
3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A; two knock- out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subge- nome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C.
[120] Such at least six knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 1 allele from the subge- nome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C.
[121] Such at least seven knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C.
[122] Such at least seven knock-out mutant Rca alleles in Brassica may also be two knock-out mu- tant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca
1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock- out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the sub- genome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca
2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock- out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the sub- genome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock- out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 2 allele from the sub- genome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock- out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the sub- genome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock- out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the sub- genome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock- out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the sub- genome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock- out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the sub- genome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock- out mutant Rca 1 allele from the subgenome A.
[123] Such at least seven knock-out mutant Rca alleles in Brassica may even be two knock-out mu- tant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subge- nome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the sub- genome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subge- nome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock- out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the sub- genome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subge- nome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock- out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the sub- genome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subge- nome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the sub- genome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A.
[124] Such at least eight knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subge- nome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock- out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subge- nome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C.
[125] Such at least eight knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A. [126] Such at least eight knock-out mutant Rca alleles in Brassica may furthermore be two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome A, two knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C andtwo knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C andtwo knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C andtwo knock-out mutant Rca 2 allele from the subgenome C; two knock- out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the sub- genome A, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C.
[127] Such at least nine knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock- out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the sub- genome A, one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock- out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subge- nome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C.
[128] Such at least nine knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, two knock-out mutant Rca 3 allele from the subgenome C and one knock-out mutant Rca 2 allele from the subgenome A.
[129] Such at least ten knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subge- nome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and one knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock- out mutant Rca 3 allele from the subgenome C; one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C.
[130] Such at least ten knock-out mutant Rca alleles in Brassica may also be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock- out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the sub- genome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C.
[131] Such at least eleven knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome C and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subge- nome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome C, two knock-out mutant Rca 1 allele from the subgenome C, one knock-out mutant Rca 3 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome C; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subge- nome C; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C, one knock-out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 3 allele from the subgenome C.
[132] Such at least twelve knock-out mutant Rca alleles in Brassica may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 3 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome C, two knock-out mutant Rca 2 allele from the subgenome C and two knock-out mutant Rca 3 allele from the subgenome C.
[133] Suitable for the invention are methods for increasing the ratio of an arginine Rca a protein variants in plants comprising introducing into cells of a cotton plant at least two, at least three, at least four, at least five, at least six, at least seven or even all eight knock-out mutant Rca a alleles according to the invention.
[134] Such at least two knock-out mutant Gossypium Rca alleles may be one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome D.
[135] Such at least three knock-out mutant Gossypium Rca alleles may be one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A and one knock- out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 2 allele from the sub- genome A and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome D and one knock- out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 2 allele from the sub- genome D and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome D and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome D and one knock-out mutant Rca 1 allele from the subgenome A.
[136] Such at least four knock-out mutant Gossypium Rca alleles may be one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome D, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subge- nome A; two knock-out mutant Rca 1 allele from the subgenome D, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome D, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome D, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome D, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome D, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A and two knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome A and two knock- out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the sub- genome D and two knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome D.
[137] Such at least five knock-out mutant Gossypium Rca alleles may be two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome D, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome D, one knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome D and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome D and one knock-out mutant Rca 1 allele from the subge- nome A; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome D and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome D, two knock-out mutant Rca 2 allele from the subgenome D and one knock-out mutant Rca 1 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome D, two knock-out mutant Rca 2 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome A.
[138] Such at least six knock-out mutant Gossypium Rca alleles may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, one knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome D, one knock-out mutant Rca 2 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome D, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 1 allele from the subgenome D; two knock- out mutant Rca 1 allele from the subgenome D, two knock-out mutant Rca 2 allele from the sub- genome D, one knock-out mutant Rca 1 allele from the subgenome A and one knock-out mutant Rca 2 allele from the subgenome A; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A and two knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D and two knock-out mutant Rca 2 allele from the subgenome D; two knock- out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the sub- genome D and two knock-out mutant Rca 2 allele from the subgenome D.
[139] Such at least seven knock-out mutant Gossypium Rca alleles may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D and one knock-out mutant Rca 2 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome D and one knock-out mutant Rca 1 allele from the subgenome D; two knock-out mutant Rca 1 allele from the subgenome A, one knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D and two knock-out mutant Rca 2 allele from the subgenome D; one knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D and two knock-out mutant Rca 2 allele from the subgenome D.
[140] Such at least eight knock-out mutant Gossypium Rca alleles may be two knock-out mutant Rca 1 allele from the subgenome A, two knock-out mutant Rca 2 allele from the subgenome A, two knock-out mutant Rca 1 allele from the subgenome D and two knock-out mutant Rca 2 allele from the subgenome D.
[141] Suitable for the invention are methods for increasing the ratio of an arginine Rca a protein variants in plants comprising introducing into cells of a soybean plant at least two, at least three, at least four, at least five, or even all six knock-out mutant Rca a alleles according to the invention.
[142] Such at least two knock-out mutant soybean Rca alleles may be two knock-out mutant Rca 1 allele; two knock-out mutant Rca 2 allele, two knock-out mutant Rca 3 allele; one knock-out mutant Rca 1 allele and one knock-out mutant Rca 2 allele; one knock-out mutant Rca 1 allele andf one knock-out mutant Rca 3 allele; one knock-out mutant Rca 2 allele and one knock-out mutant Rca 3 allele. Such at least three knock-out mutant soybean Rca alleles may be one knock-out mutant Rca 1 allele, one knock-out mutant Rca 2 allele and one knock-out mutant Rca 3 allele; two knock- out mutant Rca 1 allele and one knock-out mutant Rca 2 allele; two knock-out mutant Rca 1 allele and one knock-out mutant Rca 3 allele; two knock-out mutant Rca 2 allele and one knock-out mutant Rca 1 allele; two knock-out mutant Rca 2 allele and one knock-out mutant Rca 3 allele; two knock-out mutant Rca 3 allele and one knock-out mutant Rca 1 allele; two knock-out mutant Rca 3 allele and one knock-out mutant Rca 2 allele.
[143] Such at least four knock-out mutant soybean Rca alleles may be two knock-out mutant Rca 1 allele, one knock-out mutant Rca 2 allele and one knock-out mutant Rca 3 allele; two knock-out mutant Rca 2 allele, one knock-out mutant Rca 1 allele and one knock-out mutant Rca 3 allele; two knock-out mutant Rca 3 allele, one knock-out mutant Rca 1 allele and one knock-out mutant Rca 2 allele; two knock-out mutant Rca 1 allele and two knock-out mutant Rca 2 allele; two knock-out mutant Rca 1 allele and two knock-out mutant Rca 3 allele; two knock-out mutant Rca 2 allele and two knock-out mutant Rca 3 allele. Such at least five knock-out mutant soybean Rca alleles may be two knock-out mutant Rca 1 allele, two knock-out mutant Rca 2 allele and one knock-out mutant Rca 3 allele; two knock-out mutant Rca 1 allele, two knock-out mutant Rca 3 allele and one knock- out mutant Rca 2 allele; two knock-out mutant Rca 2 allele, two knock-out mutant Rca 3 allele and one knock-out mutant Rca 1 allele. Such at least six knock-out mutant soybean Rca alleles may be two knock-out mutant Rca 1 allele, two knock-out mutant Rca 2 allele and two knock-out mutant Rca 3 allele.
[144] “reducing the expression of endogenous Rca a protein in plant cells” refers to a reduction in the amount of a functional Rca a protein produced by the cell comprising the at least one knock- out mutant Rca allele according to the invention or the second recombinant gene capable of sup- pressing specifically the expression of the endogenous Rca gene as described above, by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% as compared to the amount of the functional Rca a protein produced by the cells not comprising the at least one knock-out mutant Rca allele according to the invention or the second recombinant gene capable of suppressing spe- cifically the expression of the endogenous Rca a gene as described above.
[145] Also suitable for the invention are methods for increasing the ratio of an arginine Rca a protein variant in wheat comprising introducing into cells of the cereal plant at least two, at least three, at least four, at least five or even all six arginine Rca a alleles according to the invention, methods for increasing the ratio of an arginine Rca a protein variant in Brassica comprising introducing into cells of the Brassica plant at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or even all twelve arginine Rca a alleles according to the invention, methods for increasing the ratio of an arginine Rca a protein variant in cotton comprising introducing into cells of the cotton plant at least two, at least three, at least four, at least five, at least six, at least seven or even all eight arginine Rca a alleles according to the invention, methods for increasing the ratio of an arginine Rca a protein variant in soybean comprising introducing into cells of the soybean plant at least two, at least three, at least four, at least five or even all six arginine Rca a alleles according to the invention. The same allele combi- nations may be made with the arginine Rca a alleles as the ones that can be made with the knock- out Rca a alleles, as described above.
[146] In another aspect, a method for increasing the photosynthetic activity of a plant is provided which comprises increasing the ratio of an arginine Rca protein variant and regenerating said plant, wherein the photosynthetic activity is increased compared to a plant not comprising said increased ratio of an arginine Rca a protein variant. In a further embodiment, the ratio of an arginine Rca a protein variant is increased according to the method for increasing the ratio of an arginine Rca a protein variant in plants described herein. In further embodiments, said arginine Rca a protein var- iant is the Rca a protein variant according to the invention. When said plant is a dicotyledone, the arginine Rca a protein variant may be a variant of an Rca a protein originating from a dicotyle- donous plant. When said plant is a monocotyledone, the arginine Rca a protein variant may be a variant of an Rca a protein originating from a monocotyledonous plant. The arginine Rca a protein variant may be a variant of an Rca a protein originating from the same species or a crossable species as the plant for which the photosynthetic activity is to be increased.
[147] The photosynthetic activity of a plant can be measured by methods well known in the art (see for example Kalaji et al 2012 Photosynth Res 114:69-96 or Long and Bemacchi, JEB 2003).
[148] In yet another aspect of the invention, a method for increasing yield of a plant, such as a wheat plant, a Brassica plant, a rice plant, a soybean plant, a cotton plant or a maize plant is provided, comprising increasing the ratio of an arginine Rca a protein variant and regenerating said plant, wherein the yield is increased compared to a plant not comprising said increased ratio of an arginine Rca a protein variant. In a further embodiment, the ratio of an arginine Rca a protein variant is increased according to the method for increasing the ratio of an arginine Rca a protein variant in plants described herein. In further embodiments, said arginine Rca a protein variant is the arginine Rca a protein variant according to the invention. The yield increased may be seed yield or thousand seed weight. When said plant is a dicotyledone, the arginine Rca a protein variant may be a variant of an Rca a protein originating from a dicotyledonous plant. When said plant is a monocotyledone, the arginine Rca a protein variant may be a variant of an Rca a protein originating from a mono- cotyledonous plant. The arginine Rca a protein variant may be a variant of an Rca a protein origi- nating from the same species or a crossable species as the plant for which the yield is to be in- creased.
[149] A method for producing a plant, such as a wheat plant, a cotton plant, a brassica plant, a rice plant, a soybean plant, a maize plant, with increased photosynthetic activity and/ or an increased yield is furthermore provided, comprising increasing the ratio of an arginine Rca a protein variant as disclosed herein and regenerating said plant.
[150] Also provided is the use of the arginine Rca a protein variant according to the invention, the nucleic acid encoding an arginine Rca a protein variant according to the invention, the recombinant gene according to the invention, the recombinant gene capable of suppressing specifically the ex- pression of the endogenous Rca genes described herein or the arginine allele of a Rca a gene pro- vided herewith to increase the ratio of an arginine Rca a protein variant in plants, to increase photosynthetic activity of a plant, to increase yield of a plant or to produce a plant with increased rubisco activity and/ or yield. Such plant may be a wheat plant, a Brassica plant, a cotton plant, a rice plant, a maize plant or a soybean plant.
[151] Yield as used herein can comprise yield of the plant or plant part which is harvested, such as biomass, or seed, including seed protein content and seed oil content, seed weight (measured as thousand seed weigth), seed number. Increased yield can be increased yield per plant and/or in- creased yield per surface unit of cultivated land, such as yield per hectare.
[152] When the yield is the seed yield, the yield increase achieved with the method described herein compared to plants wherein the ratio of an arginine Rca a protein variant is not increased may be of at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9% or at least about 10%. When the yield is the seed weight, the yield increase achieved with the method described herein compared to plants wherein the ratio of an arginine Rca a protein variant is not increased may be of at least about 5%, at least about 6%, at least about 7% or at least about 8%, at least about 9% or at least about 10%.
[153] Another aspect of the invention provides a method of producing food, feed, such as meal, grain, starch, flour or protein, or an industrial product, such as biofuel, fiber, industrial chemicals, a pharmaceutical or a nutraceutical, said method comprising obtaining the plant according to the invention or a part thereof, and preparing the food, feed or industrial product from the plant or part thereof.
[154] In case of a wheat plant or other cereal plant, examples of food products include flour, starch, leavened or unleavened breads, pasta, noodles, animal fodder, breakfast cereals, snack foods, cakes, malt, pastries, seitan and foods containing flour-based sauces.
[155] Method of producing such food, feed or industrial product from wheat are well known in the art. For example, the flour is produced by grinding finely grains in a mill (see for example www.madehow.com/Volume-3/Flour.html) and the biofuel is produced from wheat straw or mix- tures of wheat straw and wheat meal (see for example Erdei et al., Biotechnology for Biofuels, 2010, 3: 16).
[156] In yet another embodiment, a method of increasing the maximal Rubisco activase activity and/or reducing the ADP inhibition of the Rubisco activase activity of a Rca a protein is provided, comprising introducing an amino acid substitution to the amino acid sequence of said Rca a protein, wherein the amino acid substitution is substituting or replacing an amino acid with an arginine at a position corresponding to position 382 of SEQ ID NO: 7, wherein the maximal Rubisco activase activity of a Rca a protein is increased compared to the maximal Rubisco activase activity of the Rubisco activase activity of a Rca a protein not comprising the amino acid substitution and/or wherein the ADP inhibition of the Rubisco activase activity of a Rca a protein is reduced compared to the ADP inhibition of the Rubisco activase activity of a Rca a protein not comprising the amino acid substitution. In a further embodiment the maximal Rubisco activase activity of the Rca a pro- tein is increased by about 10%. In yet another embodiment, the ADP inhibition of the Rubisco activase activity of the Rca a protein is reduced by 10%.
[157] Suitable for the invention are increases in maximal Rubisco activase activity of the Rca a protein comprising said amino acid substitution by at least about or about 3%, at least about or about 4%, at least about or about 5%, at least about or about 6%, at least about or about 7%, at least about or about 8%, at least about or about 9%, at least about or about 10%.
[158] Suitable for the invention are reductions in ADP inhibition of the Rubisco activase activity of the Rca a protein comprising said amino acid substitution by at least about or about 3%, at least about or about 4%, at least about or about 5%, at least about or about 6%, at least about or about 7%, at least about or about 8%, at least about or about 9%, at least about or about 10%.
[159] In addition, a method for producing an arginine Rca a protein variant is herein provided, com- prising culturing the host cell comprising the recombinant gene comprising the nucleic acid encod- ing an arginine Rca a protein variant as described above and isolating the protein produced.
[160] Said host cell expresses or over-expresses the arginine Rca protein variant of the invention.
Accordingly, said protein of the invention is produced in and isolated from the host cell. In case that the host cell produces the protein of the invention and secretes it to the surrounding media, e. g. due to a suitable signal peptide attached to the protein, isolation denotes separation of the media comprising the protein from the host cell. Said media may then be the subject of further purification steps (see below).
[161] Suitable conditions for culturing a prokaryotic or eukaryotic host are well known to the person skilled in the art. For example, suitable conditions for culturing bacteria are growing them under aeration in Luria Bertani (LB) medium. To increase the yield and the solubility of the expression product, the medium can be buffered or supplemented with suitable additives known to enhance or facilitate both. E. coli can be cultured from 4 to about 37°C, the exact temperature or sequence of temperatures depends on the molecule to be over-expressed. In general, the skilled person is also aware that these conditions may have to be adapted to the needs of the host and the requirements of the polypeptide expressed. In case an inducible promoter controls the nucleic acid of the inven- tion in the vector present in the host cell, expression of the polypeptide can be induced by addition of an appropriate inducing agent Suitable expression protocols and strategies are known to the skilled person. [ 162] Suitable expression protocols for eukaryotic cells are well known to the skilled person and can be retrieved e.g. from Sambrook, 2001.
[163] Suitable media for insect cell culture are e.g. TNM + 10% FCS or SF900 medium. Insect cells are usually grown at 27°C as adhesion or suspension culture.
[ 164] Methods of isolation of the polypeptide produced are well-known in the art and comprise with- out limitation method steps such as ammonium sulphate precipitation, ion exchange chromatog- raphy, gel filtration chromatography (size exclusion chromatography), affinity chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC, disc gel electrophoresis or immunoprecipitation, see, for example, in Sambrook, 2001.
Cells and plants
[165] Other embodiments provide a host cell, such as an E. coli cell, an Agrobacterium cell, a yeast cell, or a plant cell, comprising (a) the recombinant gene comprising a nucleic acid encoding an arginine Rca a protein variant according to the invention or the vector comprising this recombinant gene, (b) the recombinant gene capable of suppressing specifically the expression of the endoge- nous Rca a genes as described herein or the vector comprising this recombinant gene, or (c) the arginine Rca a protein variant according to the invention.
[ 166] Further embodiments provide a plant cell comprising (a) at least one knock-out Rca a allele as described herein and/or (b) at least one arginine Rca a allele according to the invention. In yet another embodiment the plant cell comprising the recombinant gene comprising a nucleic acid en- coding an arginine Rca a protein variant according to the invention or the vector comprising this recombinant gene may further comprise the recombinant gene capable of suppressing specifically the expression of the endogenous Rca a genes as described herein or the vector comprising that recombinant gene or at least one knock-out Rca a allele as described herein. The plant cell may be a cereal plant cell, a wheat plant cell, a com plant cell, a cotton plant cell, a Brassica plant cell, a rice plant cell or a soybean plant cell.
[ 167] In yet another embodiment a plant is provided that comprises the arginine Rca a protein variant according to the invention. Said plant may be a cereal plant, a wheat plant, a com plant, a cotton plant, a Brassica plant, a rice plant or a soybean plant.
[168] Other nucleic acid sequences may also be introduced into the host cell along with the described recombinant genes described herein, e. g. also in connection with the vector of the invention. These other sequences may include 3' transcriptional terminators, 3' polyadenylation signals, other un- translated nucleic acid sequences, transit or targeting sequences, selectable markers, enhancers, and operators. Preferred nucleic acid sequences of the present invention, including recombinant vectors, structural nucleic acid sequences, promoters, and other regulatory elements, are described above.
[169] In further embodiments, a plant is provided comprising any of the recombinant genes and alleles according to the invention. A further embodiment provides plant parts and seeds obtainable from the plant according to the invention. These plant parts and seeds comprise the recombinant genes or alleles described above. In another embodiment, the plants, plant parts or seeds according to the invention are wheat plants, plant parts or seeds, Brassica plants, plant parts or seeds, rice plants, plant parts or seeds, cotton plants, plant parts or seeds, soybean plants, plant parts or seeds or com plants, plant parts or seeds.
[170] “Plants” encompasses“monocotyledonous plants” and“dicotyledonous plants”.
[171] “Monocotyledonous plants”, also known as“monocot plants” or“monocots” are well known in the art and are plants of which the seed typically has one cotyledon. Examples of monocotyle- dons plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lo- lium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, triticale, spelt, einkom, emmer, durum wheat, kamut, sorghum, and maize (com).
[172] The plants according to the invention may be cereal plants. The cereal plants according to the invention may be wheat plants, rice plants or com plants.
[173] “Wheat” or“wheat plant” as used herein can be any variety useful for growing wheat. Exam- ples of wheat are, but are not limited to, Triticum aestivum, Triticum aethiopicum, Triticum Com- pactum, Triticum dicoccoides, Triticum dicoccon, Triticum dumm, Triticum monococcum, Triti- cum spelta, Triticum turgidum.“Wheat” furthermore encompasses spring and winter wheat varie- ties, with the winter wheat varieties being defined by a vernalization requirement to flower while the spring wheat varieties do not require such vernalization to flower.
[174] “Com”,“maize”“maize plant” or“com plant” as used herein can be any variety useful for growing com. The six major types of maize (Zea mays) are dent com (Zea Mays var. indentata), flint com (Zea Mays var. indurata), pod com (Zea Mays var. tunicata), popcorn (Zea Mays var. everta), flour com (Zea Mays var. amylacea), and sweet com (Zea Mays convar. Saccharata var. mgosa).
[175] Rice contains two major subspecies: short-grained japonica or sinica varieties and the long- grained indica variety. A third subspecies which is broadgrained and thrives under tropical condi- tions was initially referred to as javanica, but is now known as tropical japonica. Whenever "rice" is used herein, it will be understood that all three subspecies are included. [176] “Dicotyledonous plants”, also known as“dicot plants” or“dicots” are well known in the art and are plants of which the seed typically has two cotyledons. Examples of families of dicotyle- donous plants are Brassicaceae, Solanaceae, Fabaceae, Malvaceae.
[177] “Brassicaceae” or“Brassicaceae plant” as used herein refers to plants belonging to the family of Brassicaceae plants, also called Cruciferae or mustard family. Examples of Brassicaceae are, but are not limited to, Brassica species, such as Brassica napus, Brassica oleracea, Brassica rapa, Brassica carinata, Brassica nigra, and Brassica juncea; Raphanus species, such as Raphanus cau- datus, Raphanus raphanistrum, and Raphanus sativus; Matthiola species; Cheiranthus species; Camelina species, such as Camelina sativa; Cramhe species, such as Cramhe b ayssinica and Crambe hispanica; Eruca species, such as Eruca vesicaria; Sinapis species such as Sinapis lba a; Diplotaxis species; Lepidium species; Nasturtium species; Orychophragmus species; Armoracia species, Eutrema species; Lepidium species; and Arabidopsis species.
[178] Said Brassicaceae plant can be a Brassica plant.“ Brassica plant” refers to allotetraploid or amphidiploid Brassica napus (AACC, 2n=38), Brassica juncea (AABB, 2n=36), Brassica carinata (BBCC, 2n=34), or to diploid Brassica rapa (syn. B. campestris) (AA, 2n=20), Brassica oleracea (CC, 2n=18) ox Brassica nigra (BB, 2n=16).
[179] “Malvaceae” as used herein refers to plants belonging to the family of Malvaceae plants, also called mallows family. Examples of Malvaceae are, but are not limited to, Gossypium species, such as Gossypium hirsutum, Gossypium barbadense, Gossypium rbor aeum and Gossypium herbaceum or progeny from crosses of such species with other species or crosses between such species.
[180] “Cotton” or“cotton plant” as used herein can be any variety useful for growing cotton. The most commonly used cotton varieties are Gossypium barbadense, G. hirsutum, G. rbo areum and G. herbaceum. Further varieties include G. fric aanum and G. raimondii. Also included are progeny from crosses of any of the above species with other species or crosses between such species.
[181] The following is a non-limiting list of cotton genotypes which can be used for transformation:
Coker 312, Coker310, Coker 5Acala SJ-5, GSC25110, Siokra 1-3, T25, GSA75, Acala SJ2, Acala SJ4, Acala SJ5, Acala SJ-C1, Acala B 1644, Acala B 1654-26, Acala B 1654-43, Acala B3991, Acala GC356, Acala GC510, Acala GAM1, Acala Cl, Acala Royale, Acala Maxxa, Acala Prema, Acala B638, Acala B1810, Acala B2724, Acala B4894, Acala B5002, Acala 1517-88, Acala 1517-91, Acala 1517-95, non Acala "picker" Siokra, "stripper" variety FC2017, Coker 315, STONEVILLE 506, STONEVILLE 825, STONEVILLE 324, STONEVILLE 453, STONEVILLE 474, STONE- VILLE KC 311, STONEVILLE LA 887, STONEVILLE 4145, STONEVILLE 4288, STONE- VILLE 4498, STONEVILLE 4554, STONEVILLE 4747, STONEVILLE 4946, STONEVILLE 5032, STONEVILLE 5115, STONEVILLE 5289, STONEVILLE 5445, STONEVILLE 5458, STONEVILLE 6182, STONEVILLE 6448, Daytona, Cobalt, DP20, DP20B, DP NUCOTN 33B, DP NUCOTN 35B, DP41, DP50, DP51, DP61, DP90, DP77, DP161, DP340, DP357, DP358, DP360, DP744, DP0912, DP0920, DP0924, DP0935, DP0949, DP0920, DP1028, DP1034, DP 1044, DP1050, DP1133, DP1137, DP1212, DP1219, DP1252, DP1311, DP1321, DP1359, DP1410, DP1441, DP1454, DP5409, DP5415, DP5461, DP5690, DP5816, MON/DP 09R303, MON/DP 09R549, MON/DP 09R550, MON/DP 09R555, MON/DP 09R573, MON/DP 09R605, MON/DP 09R615, MON/DP 09R619, MON/DP 09R621, MON/DP 09R623, MON/DP 09R627, MON/DP 09R643, MON/DP 09R796, MON/DP 09R999, MON/DP 10R013, MON/DP 10R020, MON/DP 10R030, MON/DP 10R051, MON/DP 10R052, MON/DP 11R112, MON/DP 11R124, MON/DP 11R130, MON/DP 11R136, MON/DP 11R154, MON/DP 11R158, MON/DP 11R159, MON/DP 12R224, MON/DP 12R242, MON/DP 12R244, MON/DP 12R249, MON/DP 12R251, 12R254, MON/DP 13R310, MON/DP 13R348, MON/DP 13R352, MON/DP 14R1455, MON/DP 14R1456, DP Suregrow, Suregrow 125, Suregrow 248, Suregrow 404, Suregrow 501, Suregrow 1001, DES119, McN235, HBX87, HBX191, HBX107, FC 3027, CHEMBRED Al, CHEMBRED A2, CHEMBRED A3, CHEMBRED A4, CHEMBRED Bl, CHEMBRED B2, CHEMBRED B3, CHEMBRED Cl, CHEMBRED C2, CHEMBRED C3, CHEMBRED C4, CHEMBRED CB407, PAYMASTER 145, HS26, HS46, Hyperformer 44, Hyperformer HS46, SICALA, PIMA S6 ORO BLANCO PIMA, PIMA S7, HA01, HA02, HA03, HA04, HA05, HA195, HA211, HA195, HA222, White PIMA, PHY72, PHY222, PHY333, PHY339, PHY367, PHY375, PHY417, PHY427, PHY495, PHY499, PHY565, PHY575, PHY725, PHY755, PHY800, PHY802, PHY804, PHY805, PHY811, PHY830, FM5013, FM5015, FM5017, FM989, FM832, FM966, FM958, FM989, FM958, FM832, FM991, FM819, FM800, FM960, FM966, FM981, FM1320, FM1740, FM1773, FM1830, FM1845, FM1880, FM1900, FM1944, FM2007, FM2011, FM2322, FM2324, FM2334, FM2484, FM2989, FM5035, FM5044, FM5045, FM5013, FM5015, FM5017, FM5024, FM8270, FM9058, FM9160, FM9170, FM9180, FM9250 and plants with genotypes derived thereof.
[182] “Fabaceae” as used herein refers to the plant commonly known as the legume, pea, or bean family plants. Examples of Fabaceae are, but are not limited to, Glycine max (soybean), Phaseolus (beans), Pisum sativum (pea), Cicer arietinum (chickpeas), Medicago sativa (alfalfa), Arachis hy- pogaea (peanut), Lathyrus odoratus (sweet pea), Ceratonia siliqua (carob), and Glycyrrhiza glabra (liquorice).
[183] “Plant parts” as used herein are parts of the plant, which can be cells, tissues or organs, such as seeds, severed parts such as roots, leaves, flowers, pollen, etc.
[ 184] The plants according to the invention may additionally contain an endogenous or a transgene, which confers herbicide resistance, such as the bar or pat gene, which confer resistance to glufosinate ammonium (Liberty®, Basta® or Ignite®) [EP 0 242 236 and EP 0 242 246 incorpo- rated by reference]; or any modified EPSPS gene, such as the 2mEPSPS gene from maize [EP0 508 909 and EP 0 507 698 incorporated by reference], or glyphosate acetyltransferase, or glypho- sate oxidoreductase, which confer resistance to glyphosate (RoundupRcady®), or bromoxynitril nitrilase to confer bromoxynitril tolerance, or any modified AHAS gene, which confers tolerance to sulfonylureas, imidazolinones, sulfonylaminocarbonyltriazolinones, triazolopyrimidines or py- rimidyl(oxy/thio)benzoates .
[185] The plants or seeds of the plants according to the invention may be further treated with a chemical compound, such as a chemical compound selected from the following lists: Herbicides: Clethodim, Clopyralid, Diclofop, Ethametsulfiiron, Fluazifop, Glufosinate, Glypho- sate, Metazachlor, Quinmerac, Quizalofop, Tepraloxydim, Trifluralin. Fungicides / PGRs: Azoxystrobin, N-[9-(dichloromethylene)-l,2,3,4-tetrahydro-l,4-meth- anonaphthalen-5 -yl] -3 -(difluoromethyl)- 1 -methyl- lH-pyrazole-4-carboxamide (Ben- zovindiflupyr, Benzodiflupyr), Bixafen, Boscalid, Carbendazim, Carboxin, Chlormequat-chloride, Coniothryrium minitans, Cyproconazole, Cyprodinil, Difenoconazole, Dimethomorph, Dimoxystrobin, Epoxiconazole, Famoxadone, Fluazinam, Fludioxonil, Fluopicolide, Fluopyram, Fluoxastrobin, Fluquinconazole, Flusilazole, Fluthianil, Flutriafol, Fluxapyroxad, Iprodione, Iso- pyrazam, Mefenoxam, Mepiquat-chloride, Metalaxyl, Metconazole, Metominostrobin, Paclobutra- zole, Penflufen, Penthiopyrad, Picoxystrobin, Prochloraz, Prothioconazole, Pyraclostrobin, Sedax- ane, Tebuconazole, Tetraconazole, Thiophanate-methyl, Thiram, Triadimenol, Trifloxystrobin, Bacillus firmus, Bacillus firmus strain 1-1582, Bacillus subtilis, Bacillus subtilis strain GB03, Ba- cillus subtilis strain QST 713, Bacillus pumulis, Bacillus pumulis strain GB34. Insecticides: Acetamiprid, Aldicarb, Azadirachtin, Carbofuran, Chlorantraniliprole (Rynaxypyr), Clothianidin, Cyantraniliprole (Cyazypyr), (beta-)Cyfluthrin, gamma-Cyhalothrin, lambda- Cyhalothrin, Cypermethrin, Deltamethrin, Dimethoate, Dinetofuran, Ethiprole, Flonicamid, Flubendiamide, Fluensulfone, Fluopyram, Flupyradifurone, tau-Fluvalinate, Imicyafos, Imidaclo- prid, Metaflumizone, Methiocarb, Pymetrozine, Pyrifluquinazon, Spinetoram, Spinosad, Spiro- tetramate, Sulfoxaflor, Thiacloprid, Thiamethoxam, l-(3-chloropyridin-2-yl)-N-[4-cyano-2-me- thyl-6-(methylcarbamoyl)phenyl] -3 - { [5 -(trifluoromethyl)-2H-tetrazol-2-yl]methyl } - lH-pyrazole- 5 -carboxamide, l-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-
{ [5 -(trifluoromethyl)- lH-tetrazol- 1 -yl]methyl } - lH-pyrazole-5 -carboxamide, 1 - {2-fluoro-4-me- thyl-5-[(2,2,2-trifluorethyl)sulfinyl]phenyl}-3-(trifluoromethyl)-lH-l,2,4-triazol-5-amine, (1E)- N-[(6-chloropyridin-3-yl)methyl]-N'-cyano-N-(2,2-difluoroethyl)ethanimidamide, Bacillus fir- mus, Bacillus firmus strain 1-1582, Bacillus subtilis, Bacillus subtilis strain GB03, Bacillus subtilis strain QST 713, Metarhizium anisopliae F52. [186] Whenever reference to a“plant” or“plants” according to the invention is made, it is understood that also plant parts (cells, tissues or organs, seed pods, seeds, severed parts such as roots, leaves, flowers, pollen, etc.), progeny of the plants which retain the distinguishing characteristics of the parents, such as seed obtained by selfing or crossing, e.g. hybrid seed (obtained by crossing two inbred parental lines), hybrid plants and plant parts derived there from are encompassed herein, unless otherwise indicated.
[187] In some embodiments, the plant cells of the invention as well as plant cells generated according to the methods of the invention, may be non-propagating cells.
[188] The obtained plants according to the invention can be used in a conventional breeding scheme to produce more plants with the same characteristics or to introduce the same characteristic in other varieties of the same or related plant species, or in hybrid plants. The obtained plants can further be used for creating propagating material. Plants according to the invention can further be used to produce gametes, seeds, embryos, either zygotic or somatic, progeny or hybrids of plants obtained by methods of the invention. Seeds obtained from the plants according to the invention are also encompassed by the invention.
[189] “Creating propagating material”, as used herein, relates to any means know in the art to pro- duce further plants, plant parts or seeds and includes inter alia vegetative reproduction methods (e.g. air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, twin-scaling), sexual repro- duction (crossing with another plant) and asexual reproduction (e.g. apomixis, somatic hybridiza- tion).
[190] In certain jurisdictions, plants according to the invention, which however have been obtained exclusively by essentially biological processes, wherein a process for the production of plants is considered essentially biological if it consists entirely of natural phenomena such as crossing or selection, may be excluded from patentability. Plants according to the invention thus also encom- pass those plants not exclusively obtained by essentially biological processes.
[191] The sequence listing contained in in the fde named„ 190203_ST25 txt“, which is 280 kilobytes (size as measured in Microsoft Windows®), contains 112 sequences SEQ ID NO: 1 through SEQ ID NO: 112 is filed herewith by electronic submission and is incorporated by reference herein.
[192] In the description and examples, reference is made to the following sequences:
Sequences
SEQ ID NO: 1 : amino acid sequence of the TaRca 2alpha from the subgenome A SEQ ID NO: 2: nucleotide sequence of the TaRca 2alpha from the subgenome A
SEQ ID NO: 3 : amino acid sequence of the TaRca 2alpha from the subgenome A minus the signal pep- tide
SEQ ID NO: 4: nucleotide sequence of the TaRca 2alpha from the subgenome A minus the signal peptide SEQ ID NO: 5 : amino acid sequence of the TaRca 2alpha from the subgenome B
SEQ ID NO: 6: nucleotide sequence of the TaRca 2alpha from the subgenome B
SEQ ID NO: 7 : amino acid sequence of the TaRca 2alpha from the subgenome B minus the signal peptide SEQ ID NO: 8: nucleotide sequence of the TaRca 2alpha from the subgenome B minus the signal peptide SEQ ID NO: 9: amino acid sequence of the TaRca 2alpha from the subgenome D
SEQ ID NO: 10: nucleotide sequence of the TaRca 2alpha from the subgenome D
SEQ ID NO: 11 : amino acid sequence of the TaRca 2alpha from the subgenome D minus the signal pep- tide
SEQ ID NO: 12: nucleotide sequence of the TaRca 2alpha from the subgenome D minus the signal pep- tide
SEQ ID NO: 13: amino acid sequence of the TaRca 1 beta from the subgenome A
SEQ ID NO: 14: nucleotide sequence of the TaRca 1 beta from the subgenome A
SEQ ID NO: 15: amino acid sequence of the TaRca 1 beta from the subgenome A minus the signal pep- tide
SEQ ID NO: 16: nucleotide sequence of the TaRca 1 beta from the subgenome A minus the signal pep- tide
SEQ ID NO: 17: amino acid sequence of the TaRca 1 beta from the subgenome B
SEQ ID NO: 18: nucleotide sequence of the TaRca 1 beta from the subgenome B
SEQ ID NO: 19: amino acid sequence of the TaRca 1 beta from the subgenome B minus the signal pep- tide
SEQ ID NO: 20: nucleotide sequence of the TaRca 1 beta from the subgenome B minus the signal peptide SEQ ID NO: 21 : amino acid sequence of the TaRca 1 beta from the subgenome D
SEQ ID NO: 22: nucleotide sequence of the TaRca 1 beta from the subgenome D
SEQ ID NO: 23 : amino acid sequence of the TaRca 1 beta from the subgenome D minus the signal pep- tide SEQ ID NO: 24: nucleotide sequence of the TaRca 1 beta from the subgenome D minus the signal pep- tide
SEQ ID NO: 25: amino acid sequence of the OsRca alpha
SEQ ID NO: 26: nucleotide sequence of the OsRca alpha
SEQ ID NO: 27: amino acid sequence of the OsRca alpha minus the signal peptide
SEQ ID NO: 28: nucleotide sequence of the OsRca alpha minus the signal peptide
SEQ ID NO: 29: amino acid sequence of the BnRca lalpha from the subgenome A
SEQ ID NO: 30: nucleotide sequence of the BnRca lalpha from the subgenome A
SEQ ID NO: 31 : amino acid sequence of the BnRca lalpha from the subgenome A minus the signal pep- tide
SEQ ID NO: 32: nucleotide sequence of the BnRca lalpha from the subgenome A minus the signal pep- tide
SEQ ID NO: 33: amino acid sequence of the BnRca 2alpha from the subgenome A
SEQ ID NO: 34: nucleotide sequence of the BnRca 2alpha from the subgenome A
SEQ ID NO: 35: amino acid sequence of the BnRca 2alpha from the subgenome A minus the signal pep- tide
SEQ ID NO: 36: nucleotide sequence of the BnRca 2alpha from the subgenome A minus the signal pep- tide
SEQ ID NO: 37: amino acid sequence of the BnRca 3 alpha from the subgenome A
SEQ ID NO: 38: nucleotide sequence of the BnRca 3 alpha from the subgenome A
SEQ ID NO: 39: amino acid sequence of the BnRca 3 alpha from the subgenome A minus the signal pep- tide
SEQ ID NO: 40: nucleotide sequence of the BnRca 3 alpha from the subgenome A minus the signal pep- tide
SEQ ID NO: 41 : amino acid sequence of the BnRca lalpha from the subgenome C
SEQ ID NO: 42: nucleotide sequence of the BnRca lalpha from the subgenome C
SEQ ID NO: 43: amino acid sequence of the BnRca lalpha from the subgenome C minus the signal pep- tide
SEQ ID NO: 44: nucleotide sequence of the BnRca lalpha from the subgenome C minus the signal pep- tide SEQ ID NO: 45: amino acid sequence of the BnRca 2alpha from the subgenome C
SEQ ID NO: 46: nucleotide sequence of the BnRca 2alpha from the subgenome C
SEQ ID NO: 47: amino acid sequence of the BnRca 2alpha from the subgenome C minus the signal pep- tide
SEQ ID NO: 48: nucleotide sequence of the BnRca 2alpha from the subgenome C minus the signal pep- tide
SEQ ID NO: 49: amino acid sequence of the BnRca 3 alpha from the subgenome C
SEQ ID NO: 50: nucleotide sequence of the BnRca 3 alpha from the subgenome C
SEQ ID NO: 51 : amino acid sequence of the BnRca 3from the subgenome C minus the signal peptide
SEQ ID NO: 52: nucleotide sequence of the BnRca 3from the subgenome C minus the signal peptide
SEQ ID NO: 53: amino acid sequence of the GhRca 1 alpha from the subgenome A
SEQ ID NO: 54: nucleotide sequence of the GhRca 1 alpha from the subgenome A
SEQ ID NO: 55: amino acid sequence of the GhRca 1 alpha from the subgenome A minus the signal pep- tide
SEQ ID NO: 56: nucleotide sequence of the GhRca 1 alpha from the subgenome A minus the signal pep- tide
SEQ ID NO: 57: amino acid sequence of the GhRca 2 alpha from the subgenome A
SEQ ID NO: 58: nucleotide sequence of the GhRca 2 alpha from the subgenome A
SEQ ID NO: 59: amino acid sequence of the GhRca 2 alpha from the subgenome A minus the signal pep- tide
SEQ ID NO: 60: nucleotide sequence of the GhRca 2 alpha from the subgenome A minus the signal pep- tide
SEQ ID NO: 61: amino acid sequence of the GhRca 1 alpha from the subgenome D
SEQ ID NO: 62: nucleotide sequence of the GhRca 1 alpha from the subgenome D
SEQ ID NO: 63: amino acid sequence of the GhRca 1 alpha from the subgenome D minus the signal pep- tide
SEQ ID NO: 64: nucleotide sequence of the GhRca 1 alpha from the subgenome D minus the signal pep- tide
SEQ ID NO: 65: amino acid sequence of the GhRca 2 alpha from the subgenome D
SEQ ID NO: 66: nucleotide sequence of the GhRca 2 alpha from the subgenome D SEQ ID NO: 67: amino acid sequence of the GhRca 2 alpha from the subgenome D minus the signal pep- tide
SEQ ID NO: 68: nucleotide sequence of the GhRca 2 alpha from the subgenome D minus the signal pep- tide
SEQ ID NO: 69: amino acid sequence of the GmRca 1 alpha
SEQ ID NO: 70: nucleotide sequence of the GmRca 1 alpha
SEQ ID NO: 71 : amino acid sequence of the GmRca 1 alpha minus the signal peptide
SEQ ID NO: 72: nucleotide sequence of the GmRca 1 alpha minus the signal peptide
SEQ ID NO: 73: amino acid sequence of the GmRca 2 alpha
SEQ ID NO: 74: nucleotide sequence of the GmRca 2 alpha
SEQ ID NO: 75: amino acid sequence of the GmRca 2 alpha minus the signal peptide
SEQ ID NO: 76: nucleotide sequence of the GmRca 2 alpha minus the signal peptide
SEQ ID NO: 77: amino acid sequence of the GmRca 3 alpha
SEQ ID NO: 78: nucleotide sequence of the GmRca 3 alpha
SEQ ID NO: 79: amino acid sequence of the GmRca 3 alpha minus the signal peptide
SEQ ID NO: 80: nucleotide sequence of the GmRca 3 alpha minus the signal peptide
SEQ ID NO: 81 : amino acid sequence of the ZmRca alpha
SEQ ID NO: 82: nucleotide sequence of the ZmRca alpha
SEQ ID NO: 83: amino acid sequence of the ZmRca alpha minus the signal peptide
SEQ ID NO: 84: nucleotide sequence of the ZmRca alpha minus the signal peptide
SEQ ID NO: 85: amino acid sequence of the AtRca alpha
SEQ ID NO: 86: nucleotide sequence of the AtRca alpha
SEQ ID NO: 87: amino acid sequence of the AtRca alpha minus the signal peptide
SEQ ID NO: 88: nucleotide sequence of the AtRca alpha minus the signal peptide
SEQ ID NO: 89: amino acid sequence of the TaRca 2 beta from the subgenome A
SEQ ID NO: 90: amino acid sequence of the TaRca 2 beta from the subgenome B
SEQ ID NO: 91 : amino acid sequence of the TaRca 2 beta from the subgenome D
SEQ ID NO: 92: nucleotide sequence of the T-DNA PubiZm: :hpRca
SEQ ID NO: 93: nucleotide sequence of the guide RNA gl SEQ ID NO: 94: nucleotide sequence of the guide RNA g2
SEQ ID NO: 95: nucleotide sequence of the guide RNA g 13
SEQ ID NO: 96: nucleotide sequence of the guide RNA g9
SEQ ID NO: 97: nucleotide sequence of the T-DNA 35S-cab22L::AtRca-WT SEQ ID NO: 98: nucleotide sequence of the T-DNA 35S-cab22L::AtRca-K-to-R SEQ ID NO: 99: nucleotide sequence of the TaRca-WT
SEQ ID NO: 100: nucleotide sequence of the TaRca-K-to-R
SEQ ID NO: 101: nucleotide sequence of the target sequence in RCA 2 SEQ ID NO: 102: nucleotide sequence of the forward primer
SEQ ID NO: 103: nucleotide sequence of the reverse primer
SEQ ID NO: 104: nucleotide sequence of the reference probe
SEQ ID NO: 105: nucleotide sequence of the HT-19-020 forward primer SEQ ID NO: 106: nucleotide sequence of the HT-19-020 reverse primer SEQ ID NO: 107: nucleotide sequence of the NPTII forward primer
SEQ ID NO: 108: nucleotide sequence of the NPTII reverse primer
SEQ ID NO: 109: nucleotide sequence of the NPTII probe target
SEQ ID NO: 110: nucleotide sequence of the bar forward primer
SEQ ID NO: 111: nucleotide sequence of the bar reverse primer
SEQ ID NO: 112: nucleotide sequence of the bar probe target
Examples
[193] Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out ac- cording to standard protocols as described in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Cray, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Man- ual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.
Example 1 - material and methods for the in vitro determination of the ADP inhibition of Rea proteins
Recombinant Rca protein generation
[194] All Rca genes of this study were synthesised c/e novo (GENEWIZ, South Plainfield, NJ, USA) with 46 amino acids at the N-terminal corresponding to the signal peptide deleted and a 6 amino acid His-tag attached to the C-terminal. Genes were ligated into Novagen pET-23d+ vectors (Merck KGaA, Darmstadt, Germany) before being transformed into BL21(DE3) Star Escherichia coli strain following standard procedures. 0.5-1 cultures were grown in 2-1 conical flasks and induced with 0.8 mM IPTG at an OD600 of 0.8-1, then grown for a further 17-h at 20°C. Cells were lysed by sonication for 10-sec for 5-cycles at 16-microns amplitude (Soniprep 150, MSE, London, UK). Purification was performed using 5 -ml HisTrap FF columns (GE Healthcare, Amersham, UK) fol- lowing the manufacturer’s instructions. Final Rca protein was desalted into a buffer containing 20 mM Tris pH 8, 0.2 mM EDTA, 7.5 mM MgCl2, 1 mM DTT and 50 mM KC1, at a concentration of 2.0 ± 0.5 mg/ml, snap frozen and stored at -80°C until use. Rca protein concentration was deter- mined using Protein Assay Dye Rcagent Concentrate (Bio-Rad, Hercules, CA, USA) with a bovine serum albumin (BSA) standard and molar concentration calculated using the molecular masses of 50,954 and 47, 110 Da for the an ad b isoforms, respectively.
Rubisco isolation
[195] Rubisco was extracted from the leaves of Triticum es ativum CV. Fielder (wheat) grown in a greenhouse under standard physiological conditions. Leaves were harvested greater than four hours into the light period and immediately frozen in liquid N and stored at -80°C until extraction. Frozen leaf tissue was ground into a fine powder using liquid N and a mortar and pestle. While on ice, leaf powder was added to and repeatedly vortexed in an extraction buffer consisting of 100 mM Tris pH 8.0, 1 mM EDTA, 10 mM MgCl2, 2 mM DTT, 2% W/V PVPP and protease inhibitor cocktail, before being passed through a single layer of Miricloth and Lingette Gaze to remove solid matter. The sample was spun at 24,000 g for 20-min at 4°C and supernatant kept. 35% V V of saturated ammonium sulfate was added and the sample kept on ice for 30-min before re-spinning. To the supernatant 60% V/V of saturated ammonium sulfate was added dropwise and slowly stirred at 4°C for 30 min before being re-spun. The resulting pellet was suspended in a sample buffer of 100 mM Tricine pH 8.0, 0.5 mM EDTA and desalted into the same buffer using PD-10 desalting columns. 20% glycerol was added and the sample aliquoted into 50 pi volumes before being snap frozen and stored at -80°C until use. Final purity of Rca and Rubisco were high.
Rubisco activation assays
[ 196] The velocity of Rca in activating Rubisco was measured following the ADP insensitive cou- pled-enzyme spectrophotometric method of Scales et al. (Loganathan et al., 2016, Proc. Natl. Acad. Sci. 113, 14019-14024) with the following modified details. All reagents were purchased from Merck KGaA except for d-2,3-phosphoglycerate mutase which was expressed and purified as previously outlined (Scales et al., 2014, Photosynth. Res. 119, 355-365). The assay was scaled down to 100 ml reactions and measured in Coster 96-well flat-bottom polystyrene plates (Coming, NY, USA), heated to 25°C using an Eppendorf Thermomixer (Eppendorf, Hamburg, Germany). In one set of wells a reaction solution with final volume of 80 ml was added consisting of N2 sparged MiliQ H2O, 5% W V PEG-4000, 100 mM Tricine pH 8, 10 mM MgCl2, 10 mM NaHCCfi, 5 mM DTT, 2.4 U ml Enolase, 3.75 U ml Phosphoenolpyruvate carboxylase, 6 U ml Malate dehydrogen- ase, 0.2 mM 2,3-bis-Phosphoglycerate, 4 U ml d-2, 3 -phosphogly cerate mutase, 10 U ml carbonic anhydrase and 0.8 mM NADH. When ADP inhibition was not being measured an ATP regenerating system consisting of 4 mM phosphocreatine and 20 U ml creatine phosphokinase was added. ATP and ADP were added at concentrations indicated in the text. In another set of wells a final volume of 20 ml consisted of 0.25±0.05 mM of Rubisco active sites (see below for Rubisco active site quantification) added to either; 1) an activation solution (N2 sparged MiliQ H2O, 20 mM Tricine pH 8, 20 mM NaHCO3 and 10 mM MgCL2) to determine Rubisco total carbamylated activity (ECM), or 2) 4 mM of Ribulose-l,5-bisphosphate (RuBP; 99% pure) for Rubisco substrate inhibi- tion (ER). Two minutes prior to measurements 1.2-2 mM (4 ml of ~2.0 mg ml-1) of Rca was added to ER wells as a separate droplet from the Rubisco solution. Rca was not added to ER samples when measuring spontaneous baseline activity. 10-min after addition of Rubisco, the contents of the reaction solution wells were added to the Rubisco containing wells by multi-pipette and meas- urements of absorbance at a wavelength of 340 nm immediately made on an Infinate M200 Pro plate reader (TECAN, Mannedorf, Switzerland) every 15-sec over a 8-min period. Up to 10 sam- ples were assayed simultaneously. The quantification of ECM regenerated reactions by Rca per minute (mol ECM min x10-3 mol-1 Rca) was calculated by the method outlined by Loganathan et al. 2016 over the first 4-min period of measurements . The amount of Rubisco active sites added to the assay was determined from the slope of a linear regression through the data points correspond- ing to the first 60-sec of 3-Phosphoglycetic acid (3PG) product generated from ECM samples and factoring in a wheat Rubisco reaction rate constant (Kcat) of 2.1 at 25°C (Hermida-Carrera et al., 2016, Response in Crops. Plant Physiol. 171, 2549-2561).
Statistics and data analysis
[197] All data and statistical analysis was carried out using Graphpad Prism 5.0 software (GraphPad Prism Software Inc., San Diego, CA, USA) For enzyme kinetic analysis Graphpad Prism ordinary least-squares models with best fit to the data were chosen, with iteration used to determine unknown parameters of interest. For Rca velocity versus ATP substrate concentration the hill equation [E=Emax* (6)*h/(Ahaif* (<5)h] was used. It was noted that ATP dependent velocity of Rca differed when the ATP regeneration system was not included in assays. We attribute this to ADP self-inhibition as we expect hydrolysis of ATP to ADP by Rca over the duration of the assay. We accounted for this by measuring the ATP hydrolysis activity of Rca, and including the approximate 30 mM accu- mulation of Rca over the four-minute measurement period into calculations. All experiments were repeated between 3-6 times and all values and error bars presented are means and standard devia- tion (SD).
Example 2 - Results in vitro determination of the ADP inhibition of Rea pro- teins
[ 198] The Rca a and b-isoforms of wheat and rice were both highly sensitive to ADP inhibition (Fig.
1). With a fraction of 0.1 ADP/ATP (i.e. 10% of the nucleotide content being ADP) the activation velocity of Rca was more than halved relative to velocities in the absence of ADP. The sensitivity was higher for the t ahan the b isoform for both species, evident in the higher velocities of the b- isoform in both species at a fraction of 0.1 ADP/ATP and explored more thoroughly for wheat below. Despite these subtle differences the b isoform was still highly sensitive to ADP inhibition and by 0.4 ADP/ATP both isoforms had almost no activity at all.
[199] The amino-acid position 428 for the wheat TaRca2-a isoform and 432 for the wheat TaRcal- b isoform were mutated from a native lysine (K) to either an arginine (R) or glutamine (Q). The mutation to an arginine mimics a lysine residue in a deacetylated state while the mutation to a glutamine mimics a lysine residue in an acetylated state.
[200] The single-point mutations K428Q and K428R were introduced to the CTE (C terminal exten- sion) of the TaRca2-a isoform (Fig. 2). K432Q and K432R mutations were also made to the corre- sponding Lys432 in the TaRcal-b isoform. There is no corresponding lysine residue in the TaRca2- b variant. To determine the importance of these changes on nucleotide binding and catalysis, kinetic curves were generated plotting ATP substrate versus the Rubisco activation velocity of Rca (Fig. 3). For all variants of Rca an allosteric sigmoidal response of Rca activity to ATP concentration was observed, with a Hills-slope ranging from 1.8 to 2.8, but no significant difference between any of the variants (Table 2). The ATP dependent maximum velocity of Rubisco activation (Vmax) was determined only at physiologically relevant concentrations to a maximum of 800 mM ATP. Of further note, the Vmax obtained in this study only relates to the standardized conditions under which the Rca was assayed. Preliminary experiments demonstrated that inhibited Rubisco (ER) was not at saturating substrate concentrations for Rca, but to increase ER substrate would have sped the reaction above the detection range of the spectrophotometric assay, and to use less Rca in assays was not viable due to the need for concentration dependent self-association of the Rca enzyme.
[201 ] The K428Q mutant had a significantly slower and K428R faster Vmax than the TaRca2-a wild type (Fig. 3A, Table 2). The ATP substrate concentration which corresponded with half maximal velocity (Khalf ). an indication of ATP affinity, was significantly reduced for all TaRca2-a mutations. K428Q was intermediate between the TaRca2-a wild type and K428R variant. In other words, the K428Q mutant Rca results in a reduced activity and increased ATP affinity of the Rubisco enzyme compared to a corresponding wild type Rca, while the K428R mutant Rca results in an increased activity and increased ATP affinity of the Rubisco enzyme compared to a corresponding wild type Rca. The K428R mutant Rca results in an increased ATP affinity of the Rubisco enzyme compared to the K428Q Rca mutant.
[202] A comparison of the wheat b isoform variants of Rca shows that despite the TaRca2-b isoform of wheat being sensitive to ADP inhibition it was less so than the isof aorm spliced variant, evident in it having a significantly lower Khalf than TaRca2-a (Fig. 3B, Table 2). The TaRca1-b variant, coded by a separate gene and with substantial genetic differences from the Rca2 spliced variants, had even more affinity for ATP with a Khalf significantly less than both Rca2 and Rc aa2 b isoforms and not significantly different from the TaRca2-a K428R mutant. TaRca1-b had a significantly slower Vmax than the TaRca2 spliced variants. Glutamine or arginine mutations at Lys432 of the TaRca1-b C-terminal, which correspond to the Lys428 residue of TaRca2-a, did not have any sig- nificant effect on Vmax or Khalf relative to the TaRca1-b wild type.
[203] As with Khalf . there were significant differences in ADP inhibition of Rca between the wild type isoforms and mutant variants (Fig. 4; Table 2). The TaRca2-a isoform was most susceptible to ADP inhibition with an ADP inhibitor concentration in at which half inhibitor free velocity was reached (IC50) significantly below all other variants studies (Fig. 4A, C). The order of reduced ADP inhibition, represented by increasing IC50 values, followed a similar pattern to the reduction in Khalf among the TaRca2-a variants. As such, we plotted IC50 against Khalf values and obtained a scatterplot which showed a highly significant negative linear correlation between IC50 and Khalf values, indicating that an increase in apparent affinity for ATP (i.e. reduced Khalf ) was associated with a reduction in ADP inhibitor sensitivity (i.e. increased IC50 ) (Fig. 4C). As with Khalf values, the IC50 of TaRca1-b did not significantly differ from its mutant variants and TaRca2-h had an intermediate IC50 among the wild type isoforms (Fig. 4D). Thus, both K428Q and K428R mutant Rca proteins result in a reduced ADP inhibition sensitivity of the Rubisco enzyme compared to a corresponding wild type Rca, with the K428R mutant Rca further resulting in a reduced ADP inhi- bition sensitivity of the Rubisco enzyme compared to the K428Q Rca mutant.
Figure imgf000107_0001
[204] To explore the interaction between nucleotide binding and ADP inhibition further, ATP sub- strate kinetics at differing ADP inhibitor concentrations were performed between TaRca2-a and its most ATP/ADP divergent mutant, K428R (Fig. 5). Ordinary least-squares model fitting analysis of the kinetic response to ATP and ADP allowed for the determination of the inhibitor mechanism (i.e. competitive versus non-competitive) and the apparent inhibition binding constant (¾) of ADP was obtained. A competitive-inhibition model provided a very good fit with a global R2 of > 0.98 for both variants. There was no significant difference in K, values of ADP between the TaRca2-a and K428R mutant with calculated values of 4.9±1.5 mM for TaRca2-a and 3.4±1.4 mM for K428R.
Example 3 - Generation of wheat plants with reduced level of endogenous Rca a proteins
Generation of constructs silencing specifically the endogenous Rca genes
[205] Using standard recombinant DNA techniques, the constitutive promoter region of the Ubiqui- tin gene of Zea mays according to the sequence from nucleotide position 157 to 2153 of SEQ ID NO: 92, the hairpin DNA fragment targeting the Rca genes from the subgenomes A, B and D of wheat according to the sequence from nucleotide position 2162 to 3543 of SEQ ID NO: 92, and the 3 ' untranslated sequence of the 35 S transcript gene of Cauliflower mosaic vims according to the sequence from nucleotide position 3547 to 3771 of SEQ ID NO: 92 were assembled in a vector which contains the bar selectable marker cassette (position 3856 to 5520 of SEQ ID NO: 92) to result in the T-DNA PubiZm::hpRca (SEQ ID NO: 92).
Generation of transgenic wheat plants comprising the above-mentioned silenc- ing construct
[206] The recombinant vector comprising the expression cassettes PubiZm: :hpRca is used to stably transform wheat using the method described in Yuji Ishida et al. 2015, Methods in Molecular Bi- ology, 1223: 189-198. Homozygous and null segregant plants are selected.
Generation of knock-out Rca a mutant wheat plants
By mutagenesis
[207] A mutagenized wheat population was constructed by EMS mutagenesis. Based on sequencing of the region around the Rca genes, mutant plants with a knock-out mutation in the Rca gene from either the B subgenome, from the A subgenome or from the D subgenome are identified. The ho- mozygous mutant plants and their wildtype segregants are retrieved.
[208] Such mutant plants are crossed to produce double mutant plants with a knock-out mutation in the Rca gene from both the subgenome A and B, or from both the subgenome A and D or from both the subgenome B and D. Such resulting double mutant plants are further crossed to produce mutant plants with a knock-out mutation in the Rca gene from all three subgenomes (namely A, B and D).
By targeted knock-out
[209] Guide RNAs for CRISPR-mediated gene editing targeting the mRNA coding sequence, pref- erably the protein coding sequence of the Rca gene from the D subgenome, targeting the mRNA coding sequence, preferably the protein coding sequence of the Rca genes from both the D and the A subgenomes, targeting the mRNA coding sequence, preferably the protein coding sequence of the Rca genes from both the A and the B subgenomes, or targeting the mRNA coding sequence, preferably the protein coding sequence of the Rca genes from both the A, the B and the D subge- nomes were designed by using e.g. the CAS-fmder tool. The guide RNAs were tested for targeting efficiency by PEG-mediated transient co-delivery of the gRNA expression vector with an expres- sion vector for the respective nuclease, e.g. Cas9 or Cpfl, under control of appropriate promoters, to protoplasts of a wheat line containing the Rca genes. Genomic DNA was extracted from the protoplasts after delivery of the guide RNA and nuclease vectors. After PCR amplification, integ- rity of the targeted Rca gene sequence was assessed by sequencing.
[210] The most efficient guide RNAs were used for stable gene editing in wheat. The selected guide RNAs are gl (SEQ ID NO: 93) targeting the subgenomes A, B and D; g2 (SEQ ID NO: 94) targeting the subgenomes A and D; g 13 (SEQ ID NO: 95) targeting the subgenomes A and B; and g9 (SEQ ID NO: 96) targeting the subgenome D. For this purpose, the selected guide RNA expres- sion vector, together with a nuclease expression module and a selectable marker gene, were intro- duced into wheat embryos using e.g. particle gun bombardment. Transgenic plants showing re- sistance to the selection agent were regenerated using methods known to those skilled in the art. At least 12 transgenic TO plants containing gene targeting events, preferably small deletions or inser- tions resulting in a non-functional Rca gene were identified by PCR amplification and sequencing. Examples of knock-out mutant plants obtained are shown is table 3.
[211] Table 3 Mutant lines containing TaRCA2 gene edited insertions/deletions
Figure imgf000109_0001
Figure imgf000110_0001
[212] Transgenic TO plants containing a knock-out mutation of at least one of the Rca genes, pref- erably in homozygous state, but alternatively in heterozygous state, were crossed to wild-type plants to produce T1 plants in order to eliminate integrated cas9 containing constructs. Clean T1 plants with knock-out mutations in the Rca gene from subgenome A, B and D were obtained and selfed to produce T1S1 seed. From these seeds T1S1 plants were grown that were homozygous for various knock-out mutations and selfed to produce T1S2 seed. Via this process we obtained 4 dif- ferent knock-out versions: a. version 1 : seedlot homozygous for the 4A03 mutation and a corresponding wild-type seed- lot; b. version 2: seedlot homozygous for the 4B03 mutation and a corresponding wild-type seed- lot; c. version 3: seedlot homozygous for the 4A02 and 4D03 mutation and a corresponding wild- type seedlot; d. version 4: seedlot homozygous for the 4B04 and 4D02 mutation and a corresponding wild- type seedlot.
Example 4 -Generation of wheat plants comprising an arginine Rea a pro- tein variant
[213] An adenosine deaminase base editor (ABE7.10 Gaudelli et al. 2017) was used for the intro- duction of the K428R mutation in RCA2 in wheat. The sgRNA vector comprises a cassette for the expression of the gRNA that guides the base editor for the introduction of the desired base substi- tution(s) [Lys=AAA to Arg=AGA or AGG] at the target sequence GCAGGTAAAGGGG- CACAGCA-AGG (SEQ ID NO: 101).
[214] Immature embryos, 2-3 mm size, were isolated from sterilized ears of wheat cv. Fielder and bombarded using the PDS-1000/He particle delivery system. Plasmid DNA of the the ABE7.10 and the gRNA vectors were mixed with the plasmid DNA pIB26. The vector pIB26 contains an cgfp-bar fusion gene. Bombarded immature embryos were moved to PPT containing selection media, and PPT resistant calli were selected and transferred to regeneration media for shoot for- mation.
[215] All plants developed from one embryo were treated as a pool. Genomic DNA was extracted from pooled leaf samples and the Bio-Rad QX200TM Droplet Digital PCR (ddPCR) was used to quantitatively assess the frequency of edits [Lys=AAA to Arg=AGA or AGG] . A ddPCR as- say was performed using the primer pair (Forward 5’: CTCATCAGATCGTCCAAAACAA, SEQ ID NO: 102), Reverse 5’: TCGAAGTTCTTGGCAGTCT, SEQ ID NO: 103) and the reference probe 5ΉEC- CACAGCAAGGTACTTTGCCTGT, SEQ ID NO: 104) to detect the presence of any RCA2 homoeallele and the edit probe 5’ FAM-CC+T+C+TA+C+C+TG or the edit probe 5’ FAM-CC+C+C+CTACCTG to amplify RCA2 homoeoalleles with the desired edit AAA >AGA or AAA > AGG, respectively. The’+’ preceeding the nucleotide refers to the presence of an LNA base (locked nucleic acid base).
[216] In 5 out of 284 pooled leaf samples the AGA edit was detected at a frequency between 1.1- 5.7%. Pooled leaf samples with the AGG edit were not detected. We further confirmed the pres- ence of the AGA edits in these pooled leaf samples by deep sequencing. The region surrounding the intended target site was PCR amplified with Q5 High-Fidelity polymerase (M0492L) using primer pair HT-19-020 Forward 5’: ACTTTCAGCACCTACTTTACATAC (SEQ ID NO: 105) and Reverse 5’: AGCAATCGCTTGGATGCG (SEQ ID NO: 106) amplifying a 415 bp fragment. We assessed editing frequency by calculating the percentage of sequence reads showing evidence for the presence of only the desired mutation AAA>AGA, as a proportion of the total number of reads. The edit frequencies determined by NGS analysis did correspond very well with those de- termined by ddPCR.
[217] From the pooled leaf samples where edits were detected by ddPCR; individual shoots were re- analyzed by ddPCR to detect the presence of edits in individual plantlets.
[218] As each plantlet pool is derived from a single immature embryo, all plantlets derived from a single immature embryo (plantlet pool) are considered as an independent edited event, although we can’t exclude that there might be multiple independent edited events between individual shoots derived from a single immature embryo scored as positive in the 2nd dd PCR. In a subset of the individual shoots from those pooled leaf samples, edits could be detected by dd PCR at a frequency between l . l%-20.2%. In 4 of the 5 pooled leaf samples scored as positive in the 1st edit ddPCR, individual shoots with an edit frequency of ~10 to 20% were observed indicating that one out of the 6 alleles might be precisely edited.
[219] Three plants were identified that have one precisely edited (Lys=AAA to Arg=AGA) Rca2 allele on a different subgenome (A, B & D) and no other undesired edits were transferred to the glasshouse. The three plants were crossed with wild-type plants to obtain T1 seed. T1 seed was germinated and genotyped using subgenome specific KASP assays for the AAA to AGA gene edit. T1 plants containing the AGA mutation on the A subgenome were crossed with T1 plants contain- ing the AGA mutation on the D subgenome. Progeny of the cross is genotyped and plants contain- ing both the A and D subgenome edits are selfed to obtain homozygous double mutants and wild- type segregants.
[220] The double heterozygous plants were also crossed to plants containing the B subgenome AGA mutation in order to obtain mutant plants with the AGA gene edit on all three subgenomes. Using the subgenome specific KASP assays individuals were identified that are heterozygous for the AGA gene edits on all 3 subgenomes. These plants were subsequently selfed. Progeny of the selfed triple heterozygote AGA gene edits was genotyped using the KASP assays to identify wild-type plants and triple homozygous mutants and seed was harvested from these individuals.
[221 ] In addition, double heterozygous plants were also crossed to plants contain the 4B03 knock- out on the B subgenome from Table 3. Using the KASP assays for: (i) the A subgenome AGA edit, (ii) the D subgenome AGA edit and (iii) the B subgenome 2 nucleotide deletion, individuals were identified that are heterozygous for the AGA gene edits on the A and D subgenome and het- erozygous for the 2 nucleotide deletion on the B subgenome, These plants were subsequently selfed and, using the same KASP assays as above, plants were identified that are wild-type and plants that are homozygous for the A and D subgenome AGA mutations and homozygous for the B subgenome 2 nucleotide deletion. The latter plants effectively produce 100% RCA2 protein con- taining the Lys -AAA to Arg- AGA mutation.
[222] To assess the effect of the K428R mutation in planta (in one of the three subgenomes) we used a set of events that are homozygous for the B subgenome Lys-AAA to Arg-AGA mutation and wild type for A and D subgenome, as well as events that are homozygous for B subgenome muta- tion, wild type for the A subgenome and hemizygous for D subgenome. Those plants were grown in a greenhouse. At the late tillering stage leaves are harvested and immediately frozen in liquid nitrogen and kept at -80°C until analyzed with the Rca ATPase hydrolysis assay.
[223] The Rca ATPase hydrolysis assay is carried out as described by Scafaro et al. 2016.
Example 5 -Generation of Arabidopsis plants comprising an arginine Rca a protein variant
Upscaling seeds insertion mutant
[224] Arabidopsis seeds containing a T-DNA insertion in RCA (Rubisco Activase - AT2G39730) was obtained from Nottingham Arabidopsis Stock Centre (NASC). SALK-118831 is segregating T3 plants, with chlorotic phenotypes (Columbia-0 background, T-DNA insert (NPTII gene, from pROK2)). A more detailed description of the mutant can be found in http://www.plantphys- iol.org/content/155/2/751.
[225] Arabidopsis seeds from NASC were vapor-phase sterilized for 1-2 hours according to http://plantpath.wisc.edu/afb/vapster.html.
[226] Open microfuge tubes were placed in an approximately glass desiccator container (under a fume hood) containing a beaker with 100 mL bleach (18°); 3.3 mL of glacial hydrochloric acid was then carefully added to the bleach and the jar was quickly sealed. Seed remained in contact with chlorine gas for several l-2hours and subsequently sown on petridishes containing GM2 medium [2,15 g/1 MS salts without vitamins; 1,2 % glucose; 8 g Plant agar, 112mg/l B5 vitamins].
[227] Plates with seeds were placed at 6°C for 3 days, then transferred to growth rooms (16h day/8h night, 22.5°C) for 15-20 days.
[228] All viable plants were transferred to soil in 51 well trays. DNA was extracted from leaves using standard procedures. A copy number assay for the NPTII gene [FORWARD primer 5’- ggaa- gcggtcagcccat-3’, SEQ ID NO: 107, REVERSE primer 5’- agcgttggctacccgtgata -3’, SEQ ID NO: 108, and probe targeted to the NPTII sequence FAM 5'- cgccgccaagctcttcagcaa -3' TAMRA, SEQ ID NO: 109] was used to assess the presence of the insert. Seeds were collected by using Arasystem cylinders (https ://arasystem .com/) .
Selection of heterozygous plants for floral dip
[229] Seeds harvested from individual plants carrying the NPTII insert were sown on plates with GM2 medium containing 20 mg/L kanamycin (Km, kanamycin sulfate; Duchefa).
[230] Seedlots that segregated 75% Km resistant (HH and Hh) seedlings were considered to be the result of a heterozygous parent for the T-DNA insert. Homozygous seedlings (HH) were stunted, yellow and unable to set seed. Heterozygous plants (Hh) looked green and healthy and were trans- ferred to soil (DCM potgrond voor professionals - https://dcm-info.nl/hobby/producten/potgrond, with addition of Osmocote - Osmocote Exact Standard 5-6M (15-9-2MgO; 15-3.9-10.0-1.2Mg - http : // www . icl -group . com/) .
[231] Growth conditions: 16h light/8h dark. Temperature day: 22°C, night: 20°C. Relative humidity:
60%.
Generation of constructs containing Rcaa protein variants
[232] Using standard recombinant DNA techniques, a fragment of the promoter region from the Cauliflower Mosaic Virus 35S transcript according to the sequence from nucleotide position 3006 to 3533 of SEQ ID NO: 97, the leader sequence from the chlorophyll a/b binding protein of Petunia according to the sequence from nucleotide position 3537 to 3596 of SEQ ID NO: 97, coding se- quence of rubisco activase from Arabidopsis according to sequence from nucleotide position 3603 to 5027 of SEQ ID NO 97 and a fragment of the 35S transcript gene of Cauliflower mosaic virus according to the sequence from nucleotide position 5041 to 5265 of SEQ ID NO: 97 were assem- bled in a vector which contains the bar selectable marker cassette (position 190 to 2705 of SEQ ID NO: 97) to result in the T-DNA 35S-cab22L::AtRca-WT (SEQ ID NO: 97).
[233] Using standard recombinant DNA techniques, a fragment of the promoter region from the Cauliflower Mosaic Virus 35S transcript according to the sequence from nucleotide position 3006 to 3533 of SEQ ID NO: 98, the leader sequence from the chlorophyll a/b binding protein of Petunia according to the sequence from nucleotide position 3537 to 3596 of SEQ ID NO: 98, coding se- quence of rubisco activase from Arabidopsis according to the sequence from nucleotide position 3603 to 5027 of SEQ ID NO:98, in which AAA (lysine) is mutated to AGA (argenine) by changing 1 nucleotide at position 4915 in SEQ ID NO: 97 and a fragment of the 35 S transcript gene of Cau- liflower mosaic virus according to the sequence from nucleotide position 5041 to 5265 of SEQ ID NO: 98 were assembled in a vector which contains the bar selectable marker cassette (position 190 to 2705 of SEQ ID NO: 98) to result in the T-DNA 35S-cab22L::AtRca-K-to-R (SEQ ID NO: 98).
[234] Using standard recombinant DNA techniques, a vector was assembled that contains the se- quence of SEQ ID NO: 97 where the coding sequence of the rubisco activase from Arabidopsis (nucleotide position 3603 to 5027) was replaced by the coding sequence of wheat rubisco activase (SEQ ID NO: 99) to result in the T-DNA 35S-cab22L: :TaRca-WT.
[235] Using standard recombinant DNA techniques, a vector was assembled that contains the se- quence of SEQ ID NO: 97 where the coding sequence of the rubisco activase from Arabidopsis (nucleotide position 3603 to 5027) was replaced by the coding sequence of wheat rubisco activase (SEQ ID NO: 100) in which AAA (lysine) was mutated to AGA (argenine) by changing 1 nucleo- tide at position 1283 in SEQ ID NO:99 to result in the T-DNA 35S-cab22L: :TaRca-K-to-R.
Floral dip of heterozygous Rcaa mutants
[236] The floral dip procedure (modified protocol from Clough and Bent, Plant Journal: 16:735-736, 1998. https://doi.Org/10.1046/j .1365-313x.1998.00343.x) was applied to heterozygous Rcaa plants for the four constructs described above: 35S-cab22L::AtRca-WT, 35S-cab22L::AtRca-K-to-R, 35S-cab22L: :TaRca-WT and 35S-cab22L: :TaRca-K-to-R. After harvesting seed from the dipped plants, 1 gram of seed was sown over TEKU trays (TK1520 S, Poppelmann TEKU®). Trays re- ceived a cold treatment (4°) for 2-3 days and were subsequently grown in growth rooms for 7-10 days. Seedlings were sprayed 3 times with 0,1% BASTA (150 g/1 Glyphosate - Bayer CropScience SA-NV) over an interval of 3-4 days. Surviving plantlets were transferred to soil in 51 well trays and samples were taken to test for Km copy number and bar copy number [FOREWARD primer sequence: 5’- aagcacgggaactggcat -3’(SEQ ID NO: 110) REVERSE primer: 5’- ggtaccggcaggctgaa -3’ (SEQ ID NO: 111) and probe targeted to the bar sequence: FAM 5'- ccagctgccagaaacccacgt -3' TAMRA (SEQ ID NO: 112)].
Characterisation of Arabidopsis mutants
[237] Bulked seed from individually dipped Arabidopsis mutants heterozygous for the T-DNA in- sertion were grown into plants and sprayed 3 times with basta. Surviving plants were genotyped for the T-DNA insertion in Rca and for copy number of the T-DNA containing the Arabidopsis Rca gene. Five independent plants were identified that were homozygous for the T-DNA knocking out Rca and had a single copy of the 35S-cab22L::AtRca-WT construct. Four independent plants were identified that were homozygous for the T-DNA knocking out Rca and had a single copy of the 35S-cab22L: :AtRca-K-to-R construct. [238] Seeds of three independent events carrying only the Arabidopsis Rca with the K428R mutation and three events carrying the wild type Rca were sown in 51 well sowing trays and placed in a growth chamber with a 12h light period (ca 350 PAR; constant temperature of 22°C). Leaf material was harvested for Rca ATPase hydrolysis assay from plants 3 -4 weeks after sowing. The leaf ma- terial was snap frozen in liquid nitrogen and kept at -80°C until analysis. Remaining plants are used for gas exchange measurements with a Li-6800 infra-red gas analyser system (LI-COR Inc., Lin- coln, NE, USA) connected to the whole plant Arabidopsis chamber.
[239] The ATPase hydrolysis assay is carried out according to the protocol as described in Scafaro et al. (2016). [240] Gas exchange measurements (i.e. An, net photosynthetic rates; gs, stomatal conductance for water vapor) are carried out on 3-6 plants per line and so-called‘light-induction curves’ are done as described elsewhere (Yamori et al, 2012; Scafaro et al, 2018). In short, plants are adapted to low light conditions (ca. 30 PAR) and then light intensity is raised to 1500 PAR, while gas exchange measurements are recorded every 30s until steady-state condition is reached for An. From those curves the initial linear slope is used to calculate the activation state of Rubisco. The transformation of An and linear analysis to generate the activation state constant (Ka) is the same as that outlined by Scafaro et al. (2018) and Yamori et al. (2012).
[241 ] Mixing experiments with purified Rca and Rubisco from wheat and Arabidopsis plants were performed and the results confirmed the compatibility of wheat Rca with Rubisco from Arabidop- sis. Various concentrations of Rca and Rubisco were used to verify the interactability of Rca and
Rubisco from monocot (wheat) and dicot species (Arabidopsis), which to our knowledge has not been studied so far.

Claims

Claims
1. An Rca a protein comprising an Arginine at a position corresponding to position 382 of SEQ ID NO: 7.
2. The Rca a protein according to claim 1 which comprises an amino acid sequence selected from: a. the amino acid sequence of SEQ ID NOs: 3, 7, 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87, where the amino acid at a position equivalent to position 382 of SEQ ID NO: 7 is substituted with an arginine;
b. an amino acid sequence having at least 90% identity to the amino acid sequences of SEQ ID NOs: 3, 7, 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87 and com- prising an arginine at a position corresponding to position 382 of SEQ ID NO: 7;
c. the amino acid sequences of SEQ ID NO: 3, 7, 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87, where the amino acid at a position equivalent to position 382 of SEQ ID NO: 7 is substituted with an arginine, and further comprising a chloroplast target- ing peptide;
d. an amino acid sequence having at least 90% identity to the amino acid sequences of SEQ ID NO: 3, 7, 11, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83 or 87, further comprising a chloroplast targeting peptide and comprising an arginine at a position corre- sponding to position 382 of SEQ ID NO: 7;
e. the amino acid sequences of SEQ ID NOs: 1, 5, 9, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 or 85, where the amino acid at a position equivalent to position 382 of SEQ ID NO: 7 is substituted with an arginine; or
f. an amino acid sequence having at least 90% identity to the amino acid sequences of SEQ ID NOs: 1, 5, 9, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 or 85 and compris- ing an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
3. The Rca a protein according to claim 1 or 2, which has an increased maximal Rubisco activase activity compared to an Rca a protein not comprising said arginine at a position corresponding to position 382 of SEQ ID NO: 7.
4. The Rca a protein according to any one of claims 1 to 3, which has a reduced ADP inhibition of its Rubisco activase activity compared to an Rca a protein not comprising said arginine at a position corresponding to position 382 of SEQ ID NO: 7.
5. The Rca a protein according to any one of claims 1 to 4, wherein said chloroplast targeting peptide comprises an amino acid sequence selected from: a. the amino acid sequence from position 1 to position 46 of SEQ ID NOs: 1, 9 or 25, the amino acid sequence from position 1 to position 48 of SEQ ID NO: 5, the amino acid sequence from position 1 to position 47 of SEQ ID NOs: 13, 17 or 21, the amino acid sequence from position 1 to position 59 of SEQ ID NO: 29 or 41, the amino acid sequence from position 1 to position 58 of SEQ ID NO: 33 or 45, the amino acid sequence from position 1 to position 52 of SEQ ID NO: 37 or 49, the amino acid sequence from position 1 to position 55 of SEQ ID NO: 53 or 61, the amino acid sequence from position 1 to position 51 of SEQ ID NO: 57 or 65, the amino acid sequence from position 1 to position 60 of SEQ ID NO: 69 or 77, the amino acid sequence from position 1 to position 57 of SEQ ID NO: 73, the amino acid sequence from position 1 to position 35 of SEQ ID NO: 81, or the amino acid sequence from position 1 to position 58 of SEQ ID NO: 85; and b. an amino acid sequence having at least 80% identity to the amino acid sequence from po- sition 1 to position 46 of SEQ ID NOs: 1, 9 or 25, to the amino acid sequence from position 1 to position 48 of SEQ ID NO: 5, to the amino acid sequence from position 1 to position 47 of SEQ ID NOs: 13, 17 or 21, to the amino acid sequence from position 1 to position 59 of SEQ ID NO: 29 or 41, to the amino acid sequence from position 1 to position 58 of SEQ ID NO: 33 or 45, to the amino acid sequence from position 1 to position 52 of SEQ ID NO: 37 or 49, to the amino acid sequence from position 1 to position 55 of SEQ ID NO: 53 or 61, to the amino acid sequence from position 1 to position 51 of SEQ ID NO: 57 or 65, to the amino acid sequence from position 1 to position 60 of SEQ ID NO: 69 or 77, to the amino acid sequence from position 1 to position 57 of SEQ ID NO: 73, to the amino acid sequence from position 1 to position 35 of SEQ ID NO: 81, or to the amino acid sequence from position 1 to position 58 of SEQ ID NO: 85.
6. A nucleic acid encoding the Rca a protein according to any one of claims 1 to 5 comprising a coding nucleotide sequence selected from:
a. the nucleotide sequence of SEQ ID Nos: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88, where the nucleotides encoding the amino acid at a position equivalent to position 382 of SEQ ID NO: 7 are substituted with nucleotides encoding an arginine, or the complement thereof;
b. a nucleotide sequence having at least 60% identity with the nucleotide sequence of SEQ ID Nos: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88, where the nucleotides encoding the amino acid at a position equivalent to position 382 of SEQ ID NO: 7 encode an arginine, or the complement thereof; c. the coding nucleotide sequences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88, where the nucleotides encoding the amino acid at a position equivalent to position 382 of SEQ ID NO: 7 are substituted with nucleotides encoding an arginine, or the complement thereof, and further comprising a nucleic acid encoding a chlo- roplast targeting peptide;
d. a coding nucleotide sequence having at least 60% identity to the coding nucleotide se- quences of SEQ ID NOs: 4, 8, 12, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84 or 88 and encoding an arginine at position equivalent to position 382 of SEQ ID NO: 7 and further comprising a nucleic acid encoding a chloroplast targeting peptide;
e. the coding nucleotide sequences of SEQ ID NOs: 2, 6, 10, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82 or 86, where the nucleotides encoding the amino acid at a position equivalent to position 382 of SEQ ID NO: 7 are substituted with nucleotides encoding an arginine, or the complement thereof; and
f. a coding nucleotide sequence having at least 60% identity to the coding nucleotide se- quences of SEQ ID NOs: 2, 6, 10, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82 or 86 and encoding an arginine at a position equivalent to position 382 of SEQ ID NO: 7.
7. A recombinant gene comprising the following operably linked elements:
a. a promoter, preferably expressible in plants;
b. a nucleic acid encoding the Rca a protein according to any one of claims 1 to 5 and, optionally c. a transcription termination and polyadenylation region, preferably a transcription termination and polyadenylation region functional in plants.
8. The recombinant gene according to claim 7 wherein said promoter is a constitutive promoter, tis- sue-specific promoter or an inducible promoter.
9. The recombinant gene according to claim 7 or 8, wherein said nucleic acid encoding the Rca a protein comprises the coding nucleotide sequence according to claim 6.
10. A vector comprising the recombinant gene according to any one of claims 7 to 9.
11. A host cell comprising the recombinant gene of any one of claims 7 to 9 or the vector of claim 10.
12. The cell of claim 11 which is a plant cell.
13. An allele of a Rca a gene encoding the protein according to any one of claims 1 to 5.
14. The Rca a allele according to claim 13 comprising the coding nucleotide sequence according to claim 6.
15. A plant cell comprising the protein according to any one of claims 1 to 5, the nucleic acid according to claim 6 or at least one Rca a allele according to claim 13 or 14.
16. The plant cell according to claim 12 or 15 further comprising:
a. at least one knock-out allele of an Rca a gene; or
b. a recombinant gene capable of suppressing specifically the expression of the endogenous Rca a genes.
17. The plant cell according to claim 16, wherein said recombinant gene capable of suppressing spe- cifically the expression of the endogenous Rca a genes comprises the following operably linked elements:
a. a promoter, preferably expressible in plants;
b. a nucleic acid which when transcribed yields an RNA molecule inhibitory to the endoge- nous Rca a genes encoding an Rca a protein not comprising the arginine at a position equivalent to position 382 on SEQ ID NO: 7 but not inhibitory to genes encoding the Rca a protein according to any one of claims 1 to 5; and, optionally c. a transcription termination and polyadenylation region, preferably a transcription termina- tion and polyadenylation region functional in plants.
18. The plant cell according to claim 17, wherein said promoter is a constitutive promoter, a tissue- specific promoter or an inducible promoter.
19. A plant, plant part or seed consisting essentially of the plant cells of any one of claims 12 and 15 to 18.
20. The plant, plant part or seed according to claim 19 which is a wheat plant, wheat plant part or a wheat seed.
21. The plant, plant part or seed according to claim 19 which is a soybean plant, soybean plant part or a soybean seed.
22. The plant, plant part or seed according to claim 19 which is a Brassica plant, Brassica plant part or a Brassica seed.
23. The plant, plant part or seed according to claim 19 which is a cotton plant, cotton plant part or a cotton seed.
24. The plant, plant part or seed according to claim 19 which is a rice plant, rice plant part or a rice seed.
25. The plant, plant part or seed according to claim 19 which is a com plant, com plant part or a com seed.
26. A method of increasing the maximal Rubisco activase activity of an Rca a protein comprising introducing an amino acid substitution to the amino acid sequence of said Rca a protein, wherein the amino acid substitution is a substitution of an amino acid with an arginine at a position corre- sponding to position 382 of SEQ ID NO: 7.
27. The method according to claim 26, wherein the maximal Rubisco activase activity is increased by about 10%.
28. A method of reducing the ADP inhibition of the Rubisco activase activity of an Rca a protein comprising introducing an amino acid substitution to the amino acid sequence of said Rca a protein, wherein the amino acid substitution is a substitution of an amino acid with an Arginine at a position corresponding to position 382 of SEQ ID NO: 7.
29. The method according to claim 28, wherein the ADP inhibition of the Rubisco activase activity is reduced by about 10%.
30. A method for producing the Rca a protein variant according to any one of claims 1 to 5 comprising culturing the host cell according to claim 11 and isolating the protein produced.
31. A method for increasing the ratio of the Rca a protein of claim 1 in plants comprising:
a.
i. providing to cells of a plant the recombinant gene according to any one of claims 7 to 9, and
ii. reducing the expression of an endogenous Rca a protein in said plant cells wherein said ratio is increased compared to a control plant cell not comprising said recom- binant gene; or b. introducing into cells of a plant at least one Rca a allele according to claim 13 or 14, wherein said ratio is increased compared to a control plant cell not comprising said Rca a allele.
32. The method according to claim 31, wherein reducing expression of an endogenous Rca a protein comprises:
a. introducing into said cells of a plant at least one knock-out mutant Rca allele; or b. providing said cells of a plant with a second recombinant gene capable of suppressing spe- cifically the expression of the endogenous Rca a genes.
33. The method according to claim 32, wherein said second recombinant gene capable of suppressing specifically the expression of the endogenous Rca a genes comprises the following operably linked elements:
a. a promoter, preferably expressible in plants;
b. a nucleic acid which when transcribed yields an RNA molecule inhibitory to the endoge- nous Rca genes encoding a Rca protein not comprising the arginine at a position equivalent to position 382 on SEQ ID NO: 7 but not inhibitory to genes encoding the Rca a protein according to any one of claims 1 to 5; and, optionally
c. a transcription termination and polyadenylation region, preferably a transcription termina- tion and polyadenylation region functional in plants.
34. The method according to claim 33, wherein said promoter is a constitutive promoter, a tissue-spe- cific promoter or an inducible promoter.
35. The method according to claim 31 to 34, wherein the endogenous Rca a genes comprise the coding nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86 or 88 or a coding nucleotide sequence having at least 60% identity with the nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,
70, 72, 74, 76, 78, 80, 82, 84, 86 or 88 and not encoding an arginine at a position corresponding to position 382 of SEQ ID NO: 7.
36. A method for increasing photosynthetic activity of a plant comprising:
a. increasing the ratio of the Rca a protein of claim 1 in a plant, or increasing the ratio of an Rca a protein of claim 1 in a plant according to any one of claims 31 to 35; b. regenerating said plant
wherein said photosynthetic activity is increased compared to a plant not comprising said increased ratio of the Rca a protein of claim 1.
37. A method for increasing yield of a plant comprising:
a. increasing the ratio of the Rca a protein of claim 1 in a plant, or increasing the ratio of an Rca a protein of claim lin a plant according to any one of claims 31 to 35;
b. regenerating said plant
wherein said yield is increased compared to a plant not comprising said increased ratio of the Rca a protein of claim 1.
38. The method according to claim 37, wherein said yield is seed yield.
39. The method according to claim 37, wherein said yield is thousand seed weight.
40. A method for producing a plant with an increased photosynthetic activity comprising:
a. increasing the ratio of the Rca a protein of claim 1 in a plant, or increasing the ratio of a Rca a protein of claim 1 in a plant according to any one of claims 31 to 35; b. regenerating said plant
wherein said photosynthetic activity is increased compared to a plant not comprising said increased ratio of the Rca a protein of claim 1.
41. A method for producing a plant with an increased yield comprising:
a. increasing the ratio of the Rca a protein of claim 1 in a plant, or increasing the ratio of the Rca a protein of claim 1 in a plant according to any one of claims 31 to 35; b. regenerating said plant
wherein said yield is increased compared to a plant not comprising said increased ratio of the Rca a protein of claim 1.
42. The method according to claim 40 or 41, wherein said plant is a cereal plant, such as wheat, or com, a cotton plant, a soybean plant, a brassica plant or a rice plant.
43. Use of the Rca a protein according to any one of claims 1 to 5, the nucleic acid encoding according to claim 6, the recombinant gene according to any one of claims 7 to 9, the vector according to claim 10 or the Rca a allele according to claim 13 or 14, to increase the ratio of the Rca a protein of claim 1 in a plant, to increase photosynthetic activity of a plant, to increase yield of a plant or to produce a plant with increased photosynthetic activity or with an increased yield.
44. A plant comprising the Rca a protein according to any one of claims 1 to 5.
45. A method of producing food, feed or an industrial product comprising
a. obtaining the plant, part thereof or seed according to any one of claims 19 to 25 and 43; and
b. preparing the food, feed or industrial product from said plant, part thereof or seed.
46. The method according to claim 45, wherein
a. the food or feed is meal, grain, starch, flour or protein; or
b. the industrial product is biofuel, fiber, industrial chemicals, a pharmaceutical or a nutraceu- tical.
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